A Sponsored Supplement to Science Breakthroughs in ... · Structural biology deciphers the essence of life from the invisible realm of an atom. Of all the biological disciplines,

Breakthroughs in structural biology: Celebrating 5 years of innovation at ICSB

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1TABLE OF CONTENTS

Introductions2 Interpretinglifefromthe

perspectiveofanatom

3 FromtheICSBdirectors

4 IntroductiontoICSB

Chapter 16 Exploringtheintricacyoflife

throughstructuralbiology:YigongShi’sLaboratoryRuixue Wan, Xiaofeng Zhang, Guanghui Yang et al.

9 TheResearchGroupofBrianKobilkaXiangyu Liu

11 TheLaboratoryofMaojunYangMaojun Yang

13 TheResearchGroupofHangShiHang Shi

14 TheResearchGroupofBailongXiaoBailong Xiao

15 TheResearchGroupofHaipengGongHaipeng Gong

Chapter 216 TheResearchGroupof

Hong-WeiWangYuanyuan Fan

18 TheResearchGroupofXuemingLiXueming Li

20 TheResearchGroupofQiangfengCliffZhangQiangfeng Cliff Zhang

22 TheResearchGroupofSaiLiSai Li

23 TheResearchGroupofYiXueYi Xue


XinquanWangXinquan Wang

25 TheResearchGroupofYeXiangYe Xiang

27 TheResearchGroupofPilongLiJing Wang

28 TheResearchGroupofHangHubertYinYing Zhang, Hang Yin

30 TheResearchGroupofXuTanXu Tan


HaitaoLiHuida Ma and Xingrun Zhang

35 TheLaboratoryofZhuchengChenZhucheng Chen

37 TheLaboratoryofChunlaiChenChunlai Chen

38 TheResearchGroupofXianyangFangXianyang Fang

39 TheResearchGroupofJiaweiWangJianwei Zeng, Jiawei Wang


JijieChaiJizong Wang

43 TheResearchGroupofSen-FangSuiShan Sun

This booklet was produced by the Science/AAAS Custom Publishing Office and sponsored by the Beijing Advanced Innovation Center for Structural Biology.Materials that appear in this supplement have not been peer-reviewed nor have they been assessed by Science. Articles can be cited using the following format: [AUTHOR NAME(S)] [CHAPTER TITLE] in Breakthroughs in structural biology: Celebrating 5 years of innovation at ICSB (Science/AAAS, Washington, DC, 2019), p. [xx-xx].Editor: Sean Sanders, Ph.D. Guest Editor: Natalie DeWitt, Ph.D. Proofreader/Copyeditor: Bob French Designer: JD HuntsingerROGER GONCALVES, ASSOCIATE SALES DIRECTOR Custom Publishing Europe, Middle East, and India [email protected] +41-43-243-1358© 2019 by The American Association for the Advancement of Science. All rights reserved. 20 December 2019

COVER IMAGE: Left: Spliceosome structure. Compliments of Yigong Shi. Right: DNA strand (©SVSHOT/SHUTTERSTOCK.COM)

Breakthroughs in structural biology: Celebrating 5 years of innovation at ICSB

A Sponsored Supplement to Science

Produced by the Science/AAAS Custom Publishing Office

Sponsored by

2 BREAKTHROUGHS IN STRUCTURAL BIOLOGY: CELEBRATING 5 YEARS OF INNOVATION AT ICSB

Interpreting life from the perspective of an atomStructural biology deciphers the essence of life from the invisible realm of an atom. Of all the biological disciplines, structural biology is perhaps the most interdisciplinary, drawing from biology, physics, chemistry, and engineering. Understanding the atomic structure of a protein or nucleic acid can reveal its fundamental mechanism of action. For instance, the solving of the double helix structure of DNA revealed the mechanism for DNA replication, which sparked a revolution in genetics and biology. Currently, discovering how key molecules such as protein cofactors, drugs, and carbon dioxide bind to a protein and influence its behavior is key to advancing research in medicine, agriculture, and bioengineering.

This supplement from Science explores recent advances in structural biology to commemorate the 5th anniversary of the founding of the Beijing Advanced Innovation Center for Structural Biology (ICSB) at Tsinghua University. The ICSB faculty is tackling the most fundamental questions in biology. For example, RNA splicing is one of the foundational processes in eukaryotic cells, yet the intricate machinery (known as the spliceosome) driving this process is still being elucidated. A prominent line of research at ICSB has focused on deciphering the structure of the exceptionally dynamic spliceosome. The insights gained from this research have markedly advanced our understanding of RNA splicing.

ICSB researchers also work on proteins important for medicine and drug discovery, such as G protein–coupled receptors; cytokines and their cell-surface receptors; mechanically activated cation channels that control senses such as touch, pain, and various physiological processes; immunomodulators such as the TOLL-like receptors; γ-secretase, which is implicated in Alzheimer ’s disease; and viral proteins that mediate replication and infection of human cells. Epigenetic mechanisms, which control the “reading” of the genetic code by the addition and removal of biochemical marks on histones and nucleic acids, are also being probed by structural approaches. This work is already providing fundamental insights into the mechanisms for gene expression and cell-fate decisions.

Many important biological processes are governed by large macromolecular and flexible complexes that cannot be solved by standard X-ray crystallography. Research at ICSB is investigates the development and improvement of methods for investigating such macromolecules, including cryo-electron microscopy and macromolecular crystallography. Techniques for probing dynamic processes are also being developed, such as single-molecule fluorescence labeling, which can detect molecular mechanisms at physiological concentrations, and force spectroscopy combined with optical tweezers, which can determine differences in free energy upon RNA folding. Harnessing computer algorithms to predict protein structure based on amino acid sequences and to explore protein dynamics using simulations, as well as 'omics approaches to characterize RNA structures in vivo on a transcriptome-wide level, are other areas of interest.

We hope you enjoy this diverse and inspiring collection of articles, with its remarkable breadth of sophisticated technologies and biological paradigms. There is no doubt that the innovations and insights gleaned from the work of ICSB will not only further our understanding of the intricate workings of the proteins and nucleic acids that are the engines of life itself, but will benefit the world by fueling innovations in medicine, agriculture, and environmental science.

Sean Sanders, Ph.D.Science/AAAS

3INTRODUCTIONS

From the ICSB directorsStructural biology is made up of two key words: structure and biology. Structural biology helps you see how cells, proteins, and nucleic acids work on a microscopic scale. Seeing is believing. The Beijing Advanced Innovation Center for Structural Biology (ICSB) has a very young and capable research team, where the average age of doctoral supervisors is 40. The center now has more than 20 independent laboratories, each of which is a world leader in its respective field. Structural biology in China has a celebrated history. During the 1960s, China’s first national-level leading research topics included synthesis, crystallization, and structure determination of bovine insulin. It was an important time in China’s scientific history. After 2000, China began to gain prominence in a range of structural biology areas, entering into the global arena as a serious contender in 2010. I have no doubt that the creation and development of ICSB will maintain the same level of high-quality research established in those early years. We will continue to write new chapters and create history.

— Professor Yigong Shi, Ph.D.Director of ICSB

Structural biology uses cutting-edge methodologies to investigate the detailed spatial organization and temporal changes in biological molecules, cells, tissues, and organisms, for the purpose of deciphering the basic principles of biological phenomena and the rules that govern life. The mission of ICSB includes: (1) Developing novel tools and methods, and using state-of-the-art techniques to study the most difficult and complex problems in structural biology and to make fundamental discoveries in life science; (2) Educating and nurturing the next generation of young scientists to develop motivation, creativity, and strong scientific skills that cultivate innovation. Our goal is to foster breakthrough findings that will open new doors and lead to new directions in biology research, so that we can more deeply understand our world and ourselves.

— Professor Hongwei Wang, Ph.D.Executive Deputy Director of ICSB

Structural biology investigates the molecular structures of biological macromolecules and how these structures and their alterations affect function. ICSB has grown into one of China's foremost research and education centers for structural biology in a short time. I hope that ICSB will be accepted as a world-class institute in the near future, not only performing outstanding, cutting-edge research, but also training the next generation of young structural biologists who will further scientific development in this field.

— Professor Xinquan Wang, Ph.D.Deputy Director of ICSB

The mission of our center is to resolve the mystery of biological structures in the diverse functional systems of life. ICSB researchers are committed to achieving the 3D visualization of biological assembly and interaction. We encourage fundamental scientific discovery as well as innovative technology development. Another outstanding pursuit of ICSB is to cultivate young talent in the field of structural biology. We envision that the new knowledge established in the center will be eventually be translated into real-world applications for the benefit of human health and our everyday life.

— Professor Haitao Li, Ph.D.Deputy Director of ICSB

ICSB was established in 2015. Our center has a highly efficient and professional administrative team, and most of the members hold Master ’s degrees or higher, with many having experience studying outside of China. The drive and determination of the management team to strive for success is echoed by the administrative team and demonstrated through team building, training, and execution. The mission of our administrative team is to create the optimal environment for our researchers and students, and to encourage them to participate in challenging and creative research projects. We also provide a supportive platform for researchers to achieve self-sustainable development for our center.

— Minhao Liu, Ph.D.Deputy Director/Office Director of ICSB

Yigong Shi

Hongwei Wang

BEIJING ADVANCED INNOVATION CENTER FOR STRUCTURAL BIOLOGY4

Introduction to ICSB“Structure determines function” is one of the most fundamental principles in life sciences. Structural biology utilizes scientific technologies to solve the structures and dynamics of biological macromolecules and to explain living phenomena, providing the most intuitive and foundational view for humans to investigate life’s mysteries. Structural biology has taken a huge leap forward due to recent rapid advances in cryo-electron microscopy (cryo-EM), among other technological breakthroughs. We anticipate that a revolution in structural biology is forthcoming.

In September 2015, the Beijing Advanced Innovation Center for Structural Biology (ICSB) was established at Tsinghua University with strong support from the Beijing municipal government. The center focuses on resolving the most pressing issues in structural biology, making novel and influential discoveries and answering fundamental biological questions. The research and technology applications generated by the center also facilitate the development of the biotechnology and pharmaceutical industry in Beijing. The current director, renowned structural biologist Yigong Shi, is an academician of the Chinese Academy of Sciences, a foreign associate of the U.S. National Academy of Sciences, and president of Westlake University in Hangzhou, China.

The center comprises 23 independent laboratories with over 40 postdoctoral fellows and 200 graduate students. Research carried out in the center covers many cutting-edge fields, including structural and functional research in biomacromolecular machines, disease-related membrane proteins, tumor suppressors, regulatory proteins in apoptosis, potential protein targets in diabetes treatment, and investigation into other important biomacromolecules.

Research achievements and progressResearch at ICSB is consistently recognized as being at a high level. With Tsinghua University as the corresponding institute. from October 2015 to June 2019, the center published 48 high-impact research papers in Nature, Science, and Cell, as well as dozens of papers in other high-profile journals. This recognition contributes to the center 's growing international influence, and more broadly to the promotion and development of structural biology within China.

Among the novel scientific discoveries achieved at the center, the 3D structure of the spliceosome, determined by Yigong Shi’s group, was a significant breakthrough with broad impact across the biological research arena. Since they first captured the high-resolution 3D structure of the spliceosome during pre-messenger RNA splicing, Shi's group has made a series of groundbreaking findings that further illuminate the structure and function of the spliceosome.

Additionally, the center has made considerable progress in resolving the structure and function of ion channels. Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials in nerve cells. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. In February 2017, Nieng Yan’s group at ICSB reported the cryo-EM structure of a putative Nav channel from the American cockroach (designated NavPaS) at 3.8-Å resolution. This is the first report of the 3D cryo-EM structure of a eukaryotic voltage-gated sodium channel at high resolution.

Other important research includes work from Maojun Yang’s group that reveals the structure of the mitochondrial respiratory complex, which allows for a better understanding of the organization and molecular workings of this complex, and points to possible targets for diseases related to cellular respiration; Sen-fang Sui’s work, recently published in Nature, which reports the first complete cryo-EM structure of the phycobilisome—a light-harvesting structure found in certain bacteria and algae—sheds light on phycobilisome assembly and energy transfer; and research from Ye Xiang’s group, published in Science, reports the mechanism of Ebola virus neutralization by two protective human antibodies, effective against the viruses that caused the outbreaks in Africa in 2014 and 2015, and critical in the development of vaccines.The center aims to continue developing innovative and transformative technologies for structural biology research, such as high-performance cryo-EM systems that include data acquisition, image processing, and the 3D reconstruction of biomacromolecules. ICSB has developed a long-term collaboration with the National Supercomputing Center in Wuxi to address the need for resource-hungry cryo-EM data analysis. This partnership, together with access to TaihuLight, the world’s fastest supercomputer, enables the center to better serve the needs of both basic research and future drug discovery.

5INTRODUCTIONS

The ICSB teamOur research team includes many outstanding researchers, including renowned biophysicist Sen-fang Sui and structural biologist Yigong Shi (see pg. 4), both principal investigators (PIs) at the center. The recipient of the 2012 Nobel Prize in Chemistry, Brian Kobilka, recently joined as an international scholar. The team also includes Chinese pioneers and leaders in structural biology. The young and creative team has an average age of just 40 years old.

To cultivate the next generation of structural biology researchers, the center has established a comprehensive training program. The Advanced Innovation Fellow program provides extensive resources that support trainees, allowing them to devote their passion and enthusiasm to research and engineering challenges. Fellows are supervised by multiple PIs and participate in or lead a range of research projects in the center. Following the two-year program (with a maximum of two terms), the most outstanding researchers may be asked to join the center as long-term senior researchers. More than 20 postdocs have been recruited into our program through this pathway since its inception, and have all made significant contributions to the field. For example, Ruixue Wan, a graduate student from Dr. Shi’s laboratory, entered the program in 2018. She was one of four winners of the Science & SciLifeLab Prize for Young Scientists that year (also the first graduate student from a mainland Chinese institute to win). She has published nine papers in Nature and Science as a first or co-first author and is a rising star in the field of RNA spliceosome structural biology. Furthermore, Chuangye Yan, who was another alumnus of the program and recognized as a “highly cited researcher” by Clarivate Analytics, is now a PI in the center.

We have also developed a career track for senior technicians. Since structural biology demands crossdisciplinary collaboration, training in the fields of computing, electronics, physics, mathematics, and biology is included in the program. We are currently recruiting high-performance computing cluster, storage, and network management engineers; C/C++ programmers; senior transmission electron microscopy technicians; technicians trained in protein expression, purification, and identification; and engineers/technicians familiar with microbiology fermentation.

Our center has invited world-famous structural biologists, senior researchers from top international institutes, and professors from prestigious universities to be international academic advisors, leading to long-term collaborations. They provide suggestions on scientific research direction, recruitment and training, and brand building. Our current international academic advisors committee includes Dr. Robert Roeder, a pioneer in eukaryotic transcription, professor at Rockefeller University, and a member of the U.S. National Academy of Sciences; Dr. Dinshaw J. Patel, a structural biologist from Memorial Sloan Kettering Cancer Center and a member of the U.S. National Academy of Sciences and the American Academy of Arts and Sciences; Dr. Robert Glaeser, a structural biologist from University of California, Berkeley; Dr. Eva Nogales from the University of California, Berkeley, a member of the U.S. National Academy of Sciences and the American Academy of Arts and Sciences; Dr. Patrick Cramer, a pioneer in RNA polymerase research and director of the Max Planck Institute for Biophysical Chemistry; Dr. Kurt Wüthrich, 2002 Nobel Prize laureate in Chemistry and nuclear magnetic resonance expert from the Scripps Research Institute; Dr. Peijun Zhang, renowned molecular biologist from Oxford University; and Dr. Joachim Frank, 2017 Nobel Prize laureate in Chemistry, who joined the committee in 2018.

Last, but not least, our center has hosted international academic conferences once or twice each year since its establishment to facilitate communication and promote scientific collaboration.


Exploring the intricacy of l i fe through structural biology: Yigong Shi ’s LaboratoryRuixue Wan1, Xiaofeng Zhang2, Guanghui Yang1, Yini Li1, Yigong Shi1,2*

Yigong Shi received his bachelor ’s degree in biology with highest honors from Tsinghua University in 1989. For his graduate dissertation, he studied zinc-finger proteins under the guidance of Jeremy M. Berg and received his doctorate in biophysics from Johns Hopkins University in 1995. He pursued postdoctoral training in Nikola P. Pavletich’s laboratory at the Memorial Sloan Kettering Cancer Center where he worked on Smad proteins in transforming growth factor beta (TGF-β) signaling. Shi joined the Department of Molecular Biology at Princeton University as an assistant professor in 1998 and became a tenured professor in 2003. He became a Warner-Lambert/Parke-Davis Professor in 2007. A year later, Dr. Shi resigned from Princeton University, declined an offer to be an Investigator at the Howard Hughes Medical Institute, and returned to Tsinghua University, where he has led the development of a biomedical research community for a decade. In April 2018, Shi was elected as the founding president of Westlake University, the first private research university in China.

The Shi Laboratory at Tsinghua University combines structural biology, biochemistry, and biophysics to investigate the molecular and chemical basis of fundamental cellular processes, particularly spliceosome-catalyzed pre-messenger RNA (pre-mRNA) splicing, regulated intramembrane proteolysis, and caspase-dependent cell death. For his contributions, Shi has received several awards, including the 2003 Irving Sigal Young Investigator Award from the Protein Society, the 2010 Raymond and Beverly Sackler International Prize in Biophysics, the 2014 Gregori Aminoff Prize from the Royal Swedish Academy of Sciences, and the 2017 Future Science Prize in life sciences. He is a member of the Chinese Academy of Sciences, an Honorary Foreign Member of the American Academy of Arts and Sciences, a Foreign Associate of the U.S. National Academy of Sciences, and a Foreign Associate of the European Molecular Biology Organization. Shi’s laboratory has made many important contributions to structural biology, as summarized below.

1. Elucidation of the structural basis of pre-mRNA splicing by yeast and human spliceosomes

According to the central dogma of molecular biology, information flows from genes to proteins. In all eukaryotes, this flow comprises three steps: transcription of DNA into pre-mRNA, splicing of pre-mRNA into mRNA, and translation of mRNA into protein. Pre-mRNAs often contain multiple exons, each flanked by two noncoding introns of varying lengths. Pre-mRNA splicing, through intron excision and exon ligation, increases the complexity of eukaryotic gene expression (1, 2). Aberrant splicing contributes to approximately one-third of human genetic disorders (3).

Pre-mRNA splicing is catalyzed by the spliceosome, a supramolecular ribonucleoprotein (RNP) complex that undergoes assembly, activation, catalysis, and disassembly in each splicing cycle (4–7). The spliceosome comprises five small nuclear RNPs

(snRNPs) (U1, U2, U4, U5, and U6 snRNPs) (8) and various associated proteins. Eight major functional states of the assembled spliceosome have been reported (9–11): the precursor precatalytic spliceosome (pre-B), precatalytic spliceosome (B), activated spliceosome (Bact), catalytically activated spliceosome (B*), step I spliceosome (C), step II activated spliceosome (C*), postcatalytic spliceosome (P), and the intron lariat spliceosome (ILS). The splicing reaction comprises two sequential transesterification steps—branching and exon ligation—which are catalyzed by the B* and C* complexes, respectively.

Since 2008, Shi’s laboratory has focused on the structural elucidation of the spliceosome. Based on preliminary findings regarding certain spliceosomal components (12, 13), Shi’s group determined the first near-atomic structure of an intact spliceosome

FIGURE 1. Structure of the intron lariat spliceosome from Schizosaccharomyces pombe at 3.6-Å resolution. Reprinted from (24), with permission from Elsevier.

FIGURE 2. A complete cycle of spliceosomal pre-messenger RNA (pre-mRNA) splicing. Cryo-electron microscopic structures of the yeast spliceosome shown here were previously determined by Shi’s group.

1�Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�China2School�of�Life�Sciences,�Westlake�University,�Hangzhou,�Zhejiang,�China*Corresponding�author:�[email protected]

CHAPTER 1 7

of the ILS complex from Schizosaccharomyces pombe at an average resolution of 3.6 Å in 2015 (14, 15) (Figure 1). This structure unveiled the conserved overall organization and the conserved configuration of the active site of the spliceosome, thus providing unprecedented insights into the assembly and mechanism of action of the spliceosome. The structure unambiguously revealed that the spliceosome is a protein-directed ribozyme, with the protein components essential for the timely delivery of critical RNA elements into close proximity during the splicing reaction.

Since then, Shi’s group has elucidated the cryo-electron microscopic (cryo-EM) structures of the Saccharomyces cerevisiae spliceosome in all eight major fully assembled states: pre-B (3.3–4.6 Å) (16), B (3.9 Å) (16), Bact (3.5 Å) (17), B* (2.9–3.8 Å) (18), C (3.4 Å) (19), C* (4.0 Å) (20), P (3.6 Å) (21), and ILS (3.5 Å) (22). Furthermore, the group resolved the structure of the U4/U6.U5 tri-snRNP of S. cerevisiae (23) (Figure 2). Together, these structures reveal the mechanisms underlying spliceosomal pre-mRNA splicing.

Pre-mRNA splicing is evolutionarily conserved from yeast to humans. Nevertheless, splicing is more sophisticated in higher eukaryotes. Most human pre-mRNAs are subjected to alternative splicing, a regulated but poorly understood phenomenon. In 2017, Shi’s group reported the first near-atomic structure of the human spliceosome of the C* complex (25), which exhibits striking features, including the promotion of exon ligation (Figure 3A). Thereafter, they elucidated the structure of seven of the eight major functional

states of the human spliceosome, except the B* complex (26–29). The overall organization and active site conformation are similar between yeast and human spliceosomes. Unique structural features present only in the human spliceosome provide novel insights into the mechanism underlying alternative splicing. For example, the metazoan specific splicing factor PRKR interacting protein 1 (PRKRIP1), first detected in the human C* complex (25), may promote exon ligation by stabilizing the active site conformation (Figure 3B).

2. Structural biology of γ-secretase and its role in Alzheimer ’s disease

Alzheimer ’s disease (AD) affects over 40 million individuals worldwide and is characterized by the presence of β-amyloid plaques in the brain (30). Dysfunction of the intramembrane protease γ-secretase is thought to cause AD through preferential generation of certain β-amyloid peptides, which accumulate to form β-amyloid plaques (31, 32). Most mutations in AD patients map to presenilin 1 (PS1), the catalytic subunit of γ-secretase.

The Shi laboratory has been working on intramembrane proteases since 2004 and has determined the X-ray structures of a rhomboid family serine protease GlpG and a site-2 protease family metalloprotease S2P (33, 34). They then resolved the X-ray structures of a presenilin homolog, designated PSH (35) and the extracellular domain of γ-secretase (36). In collaboration with

FIGURE 3. Unique features of the human spliceosome. (A) The first atomic model of the human spliceosome. (B) Striking features of the metazoan-specific splicing factor PRKR interacting protein 1 (PRKRIP1). Adapted from (25). CC BY (http://creativecommons.org/licenses/by/4.0/).

FIGURE 4. Structures of human γ-secretase. (A) X-ray structure of a presenilin homolog PSH from Methanoculleus marisnigri JR1 (PDB code: 4HYG) (35). (B) X-ray structure of the extracellular domain of nicastrin from Dictyostelium purpureum (PDB code: 4R12) (36). (C) Structure of the intact human γ-secretase determined at 3.4-Å resolution (PDB code: 5A63) (41).


Sjors Scheres (Medical Research Council Laboratory, Cambridge University, United Kingdom), the Shi group elucidated the cryo-EM structure of human γ-secretase first at 4.5-Å resolution (37) and then at 3.4 Å (38), thus facilitating atomic modeling of the four components of γ-secretase and structural insights into AD-derived mutations (Figure 4). Furthermore, they elucidated the structural basis of substrate recognition by resolving the structures

of γ-secretase in complex with two important substrates, Notch and amyloid precursor protein (APP), at 2.7-Å and 2.6-Å resolution, respectively (39, 40). PS1 and the substrate both undergo prominent conformational rearrangements upon association (Figure 5). These structures provide mechanistic insights into AD pathogenesis.

3. Structural biological insights into caspase-dependent apoptosis

The Shi laboratory has been working on apoptosis since 1998. Apoptosis plays an essential role in metazoan development, and its dysregulation may result in cancer and autoimmune disorders. Initiation of apoptosis is evolutionarily conserved among metazoans (Figure 6), culminating in the activation of caspases (42). Apoptotic caspases are classified into two classes: initiator and effector caspases. Initiator caspases are activated by a multiprotein complex known as the apoptosome and are responsible for the cleavage and activation of downstream effector caspases, which execute cell death (43).

Using a structural biology approach, Shi’s group elucidated the mechanisms underlying the inhibition of apoptotic protease activating factor 1 (Apaf-1) and CED-4, the primary components of the apoptosome in mammals and Caenorhabditis elegans, respectively (44, 45); apoptosome assembly (46–49); recruitment and activation of initiator caspases (46, 50–53); activation of effector caspases (54, 55); inhibitors of apoptosis (IAP)-mediated inhibition of caspases (55); and suppression of IAP-mediated inhibition by second mitochondria-derived activator of caspases (Smac)-like proteins (55). These studies not only provide molecular and mechanistic insights into the complex network that regulates apoptosis, but also reveal evolutionary conservation and variations among the principal mechanisms in different organisms to achieve the common outcome of cell death.

PerspectiveStructural biology has entered a new era, with the routine

elucidation of transient expression states in mammalian cells and remarkable advancements in EM. Numerous previously insurmountable targets, exemplified by dynamic supramolecular

FIGURE 5. Cryo-electron microscopic structure of human γ-secretase in complex with an amyloid precursor protein (APP), fragment. (A) Structure of the human γ-secretase bound to an APP fragment (PDB code: 6IYC) (40). (B) Binding of APP induces the formation of two β-strands in PS1, which form a hybrid β-sheet with a β-strand from APP. (C) Upon binding to γ-secretase, the C-terminal helix of APP is unwound into an extended conformation to expose the cleavage sites. Adapted from (40). CC BY (http://creativecommons.org/licenses/by/4.0/).

FIGURE 6. Conserved apoptotic pathways in Caenorhabditis elegans , Drosophila, and mammals. Conserved proteins are indicated by the same color, with initiator and effector caspases indicated in magenta and blue, respectively. Activated CED-4, apoptotic protease-activating factor 1 (Apaf-1), and Drosophila Apaf-1 (dApaf-1) form the apoptosome. Inhibitors of apoptosis and the second mitochondria-derived activator of caspases (Smac)-like protein are indicated in cyan and green. Adapted from (56). Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society.

CHAPTER 1 9

complexes and integral membrane proteins, have been successfully characterized at atomic resolution. Unprecedented opportunities have rapidly emerged and new challenges have been addressed. The Shi laboratory continues to use state-of-the-art methods to investigate dynamic mechanisms underlying pre-mRNA splicing, intramembrane proteolysis, and apoptosis, to elucidate the principles governing natural phenomena and to translate basic research discoveries for the welfare of humankind.

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(1907).31. J. A. Hardy, G. A. Higgins, Science 256, 184–185 (1992).32. D. R. Borchelt et al., Neuron 17, 1005–1013 (1996).33. Z. Wu et al., Nat. Struct. Mol. Biol. 13, 1084–1091 (2006).34. L. Feng et al., Science 318, 1608–1612 (2007).35. X. Li et al., Nature 493, 56–61 (2013).36. T. Xie et al., Proc. Natl. Acad. Sci. U.S.A. 111, 13349–13354 (2014).37. P. Lu et al., Nature 512, 166–170 (2014).38. X. C. Bai et al., Nature 525, 212–217 (2015).39. G. Yang et al., Nature 565, 192–197 (2019).40. R. Zhou et al., Science 363, eaaw0930 (2019).41. X. C. Bai, E. Rajendra, G. Yang, Y. Shi, S. H. W. Scheres, eLife 4, e11182 (2015).42. N. A. Thornberry, Y. Lazebnik, Science 281, 1312–1316 (1998).43. Y. Shi, Mol. Cell. 9, 459–470 (2002).44. S. J. Riedl, W. Li, Y. Chao, R. Schwarzenbacher, Y. Shi, Nature 434, 926–933 (2005).45. N. Yan et al., Nature 437, 831–837 (2005).46. Y. Pang et al., Genes Dev. 29, 277–287 (2015).47. S. Qi et al., Cell 141, 446–457 (2010).48. M. Zhou et al., Genes Dev. 29, 2349–2361 (2015).49. N. Yan et al., Mol. Cell 15, 999–1006 (2004).50. Q. Hu et al., Proc. Natl. Acad. Sci. U.S.A. 111, 16254–16261 (2014).51. W. Huang et al., Genes Dev. 27, 2039–2048 (2013).52. Y. Li et al., Proc. Natl. Acad. Sci. U.S.A. 114, 1542–1547 (2017).53. H. Qin et al., Nature 399, 549–557 (1999).54. J. Chai et al., Cell 107, 399–407 (2001).55. N. Yan, Y. Shi, Annu. Rev. Cell Dev. Biol. 21, 35–56 (2005).56. Y. Shi, Protein Sci. 13, 1979–1987 (2004).

The Research Group of Brian KobilkaXiangyu Liu

Brian Kobilka received his bachelor ’s degree from the University of Minnesota Duluth in 1977 and his M.D. from the Yale School of Medicine in 1981. After his residency at Barnes-Jewish Hospital in St. Louis, Missouri, he worked as a postdoctoral fellow in Robert Lefkowitz’s group at Duke University, where he cloned genes encoding adrenergic receptors, notably, the beta-2 adrenergic receptor (β2AR). In 1989, Kobilka moved to Stanford and established a research group to study the molecular mechanisms underlying the function of G-protein-coupled receptors (GPCRs). He was awarded the Nobel Prize in Chemistry in 2012 with Robert Lefkowitz.

Currently, Kobilka is a professor in the Department of Molecular and Cellular Physiology at Stanford University School of Medicine. In 2012, he became a visiting professor at Tsinghua University School of Medicine, where his laboratory consists of one assistant

researcher, one staff member, three postdoctoral fellows, and six graduate students. The group focuses on GPCR structure, dynamics, and drug development. The techniques used by the group include X-ray crystallography, cryo-electron microscopy, nuclear magnetic resonance spectroscopy, single-molecule fluorescence resonance energy transfer, and structure-based drug discovery.

The main contributions of Kobilka’s research group are summarized below.

1. Identifying allosteric modulator pockets in β2ARThere is intense interest in developing agonist or antagonist

compounds to modulate GPCRs, which can occur both orthosterically and allosterically. Because orthosteric pockets are highly conserved within the same GPCR subfamily, drug development involving orthosteric modulators is often limited by poor subtype selectivity. Conversely, allosteric modulators are more likely to be selective, as their binding sites are less conserved.

In collaboration with Lefkowitz’s research group, Kobilka’s group determined the inactive β2AR structure with the orthosteric antagonist carazolol, and the first negative allosteric modulator for β2AR, Cmpd-15PA (1), as well as the active β2AR structure bound to the orthosteric agonist, BI167107, and the first positive allosteric modulator for β2AR, Cmpd-6FA (Figure 1) (2). These structures

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Pharmaceutical�Sciences,�Tsinghua�University,�Beijing,�[email protected]


provide insights into the mechanisms underlying the allosteric modulation of β2AR orthosteric binding and signaling. Furthermore, the research sheds light on the development of specific allosteric modulators of other GPCRs.

2. Uncovering the GPCR-G protein complex formation processAll known GPCR-G structures capture the nucleotide-free state

of the G protein. Several lines of evidence suggest that the GDP-bound G protein and its GPCR form an intermediate complex during G-protein activation. Starting with an effort to stabilize GPCRs in an active conformation by protein engineering, Kobilka’s research group solved the β2AR structure when the receptor was fused to the Gs protein’s C-terminal peptide (GsCT) and discovered an alternative interaction between active β2AR and GsCT. Through site-directed mutagenesis as well as functional and biophysical studies, the group provided evidence that this alternate interaction may

represent an intermediate state in the β2AR activation of Gs protein and contribute to Gs coupling specificity (3) (Figure 2). This work illuminates the dynamic processes of G-protein activation.

3. Clarifying the complex dynamics of the M2 muscarinic acetylcholine receptor conformation

Crystal structures provide static information regarding inactive and active GPCR conformations. However, little is known about the potential allosteric links between extracellular ligand-binding pockets and the intracellular transducer coupling interface. The M2 muscarinic acetylcholine receptor (M2R) regulates heart rate and is pivotal to central nervous system functions. Because of the

FIGURE 1. β2AR structure bound to different modulators . Left panel, inactive β2AR (orange) bound to orthosteric antagonist carazolol (pink) and Cmpd-15PA (blue). Right panel, active β2AR (green) bound to Gs mimic nanobody Nb6B9 (red), orthosteric agonist BI167107 (purple), and Cmpd-6FA (yellow).

FIGURE 2. Alternative interaction pattern observed between β2AR and GsCT in active β2AR fused to GsCT. Based on this alternative interaction, we proposed a model of β2AR–Gs complex formation. Reprinted from (3). Copyright 2019, with permission from Elsevier.

FIGURE 3. Conformational complexity and dynamics of the M2 muscarinic receptor. Adapted from (4). Copyright 2019, with permission from Elsevier.

FIGURE 4. Structure-guided design of the M3 muscarinic-selective antagonist based on a single amino-acid difference between M3R and M2R.

CHAPTER 1 11

existence of both orthosteric ligands and allosteric modulators that can regulate M2R, it is a model system for GPCR pharmacology.

In collaboration with Changwen Jin at Peking University, Kobilka’s research group employed solution nuclear magnetic resonance to study M2R dynamics when it was bound to multiple orthosteric and allosteric ligands. The results showed that M2R is a highly dynamic receptor with conformational plasticity, and ligands with different signaling profiles act to stabilize distinct GPCR conformations (4) (Figure 3). This work may guide the development of pharmaceuticals that regulate specific signaling pathways.

4. Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists

High sequence homology within one GPCR subfamily precludes subtype selectivity in many GPCR drugs. One example is tiotropium, an M3 muscarinic acetylcholine receptor (M3R) antagonist used to treat chronic obstructive pulmonary disease. Tiotropium has a similar affinity for M3R and M2R. Thus, developing M3R-selective antagonists may reduce the unwanted side effects associated with binding to the M2R, which regulates heart rate. To do so, Kobilka’s research group collaborated with Peter Gmeiner ’s research group

at Friedrich-Alexander University (Germany) and Brian Shoichet’s research group at the University of California, San Francisco, as well as Roger Sunahara’s research group at the University of California, San Diego. Using molecular docking and structure-based design, the researchers took advantage of a single amino-acid difference between M2R and M3R to develop a selective antagonist with 100-fold higher affinity for the latter over the former. Further, they confirmed docking-predicted geometry with a 3.1-Å crystal structure of M3R bound to the novel selective antagonist (5) (Figure 4). These findings highlight the potential of designing subtype-selective GPCR drugs based on structure.

In the future, Kobilka’s research group will continue studying GPCR with the long-term goal of developing novel GPCR drugs with improved clinical properties.

References1. X. Liu et al., Nature 548, 480–484 (2017).2. X. Liu et al., Science 364, 1283–1287 (2019).3. X. Liu et al., Cell 177, 1243–1251 e1212 (2019).4. J. Xu et al., Mol. Cell 75, 53–65 e7 (2019).5. H. Liu et al., Proc. Natl. Acad. Sci. U. S. A. 115, 12046–12050 (2018).

The Laboratory of Maojun YangMaojun Yang

Maojun Yang, professor and Ph.D. supervisor at the School of Life Sciences, Tsinghua University, is a prominent scientist in mitochondrial respiratory chain research. Since joining Tsinghua University, Yang has focused mainly on the structural study of cellular respiration–related proteins, including type-II NADH dehydrogenase (NDH2) in yeast and malarial parasites, and respiratory chain complexes and adenosine triphosphate (ATP) synthase in mitochondria. His work has formed a solid basis for elucidation of the molecular mechanisms involved in drug discovery. To date, he has published more than 70 scientific articles and is corresponding author for 40 of them. Many of these articles have been published in premier journals such as Science, Nature, and Cell. After years of hard work, he was selected as a Changjiang Scholar by the Ministry of Education and was supported by the National Science Fund for Distinguished Young Scholars in 2016. He was named as the chief scientist of the Ministry of Science and Technology’s key R&D program, “Structural Biology Research on Super Large Protein Machines,” in 2017. The major achievements of Yang’s laboratory are summarized below.

1. Structure of the mitochondrial respiratory supercomplexYang’s laboratory was the first to solve the high-resolution

cryo-electron microscopy (cryo-EM) structures of porcine (1, 2) and human (3) respiratory supercomplexes (SCs). His group clearly identified detailed interactions between individual complexes within the SC, indicating that these individual complexes function in a coordinated manner within the SC (Figure 1A). After subregion refinement, Yang’s group obtained a high-resolution structure

of the intact 14-subunit (including NDUFA4) complex IV, and provided biochemical data to support the hypothesis that the functional complex IV occurs as a monomer in vivo (4). They found that complex III is a 21-subunit asymmetric dimer (5), and also discovered a megacomplex structure that could be the highest-order assembly of respiratory complexes (Figure 1B).

Yang’s group proposed an electron transfer mechanism in complex III that is different from the popular Q-cycle theory. They consider that only the quinone reduction (Qi) site is the bona fide ubiquinol (UQH2) binding site, and only the hydroquinone oxidation (Qo) site can be blocked by inhibitors. Further, they believe that the thermodynamics of the overall reaction in Q-cycle theory that uses UQH2 to reduce ubiquinone (UQ) is not favorable. Their unpublished data revealed the presence of a UQ molecule at the Qi site, and the blockage of the Qo site by the long tail of UQ after soaking the SC sample with UQ; this is in contrast to published results in which no UQ has been detected in any available structure of mitochondrial complex III.

2. Structure of ATP synthase tetramerATP synthase is the major ATP source in most mammalian

cells. Although biochemical and structural studies have been performed on this enzyme for more than half a century, the precise arrangement of its membrane subunits and regulatory mechanism are still unclear. Yang’s group identified a tetrameric form of ATP synthase in the inhibited state and solved its high-resolution structure by cryo-EM microscopy (6). According to this structure, two (previously reported) V-shaped ATP synthase dimers form a C2 rotational, symmetrical H-shaped tetramer in an antiparallel pattern. The neighboring F1 regions are linked by antiparallel dimeric IF1 proteins. Furthermore, four C-terminal ends of the e-subunits are found to interact with four C8 rings in an intervening manner, indicating that the tetrameric ATP synthase is an integral functional unit, at least in the inhibited state (Figure 1C).

Owing to an increasing number of reports that demonstrate the influence of mitochondria-shaping proteins [including the mitochondrial contact site and cristae organizing system (MICOS),

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�[email protected]


optic atrophy 1 (OPA1), and the prohibitin (PHB) family] on the assembly and efficiency of the SC and ATP synthase dimers, it is widely accepted that the shape of cristae and the architecture of oxidative phosphorylation (OXPHOS) machinery are tightly connected. A fairly convincing model has been proposed that describes the relationship between MICOS, SCs, ATP synthase, and cristae shape. This model states that MICOS proteins localize at cristae junctions, giving rise to a negative membrane curvature; ATP synthase dimers localize at the ridge of the cristae, giving rise to a positive membrane curvature; and SCs are distributed along each side of the ATP synthase dimers (Figure 1D).

3. Structure of NDH2 and antimalarial drug screeningNDH2 functions as an alternative to multisubunit respiratory

chain complex I, which catalyzes electron transfer from NADH to UQ during cellular respiration. Ndi1 (NDH2 from yeast) is a valuable therapeutic agent, because its expression can restore the function of complex I in humans. NDH2 from Plasmodium falciparum (PfNDH2) is also worth taking into consideration while designing antimalarial drugs, as it is critical for the survival of the malarial parasite. Yang’s

group reported high-resolution crystal structures of Ndi1 and PfNDH2 in 2012 (7) and 2017 (8), respectively, which revealed the regulatory and catalytic mechanism of NDH2, thereby providing a solid basis for drug development (Figure 1E and 1F).

Yang has made major breakthroughs in the field of mitochondrial respiratory chain complexes. He not only filled in lacunae regarding individual complexes but also provided convincing ideas about the behavior of large protein assemblies. Yang’s group is now a forerunner in the field of cellular respiration–related structural studies.

References1. J. Gu et al., Nature 537, 639–643 (2016).2. M. Wu, J. Gu, R. Guo, Y. Huang, M. Yang, Cell 167, 1598–1609.e10 (2016).3. R. Guo, S. Zong, M. Wu, J. Gu, M. Yang, Cell 170, 1247–1257.e12 (2017).4. S. Zong et al., Cell Res. 28, 1026–1034 (2018).5. S. Zong et al., Protein Cell 9, 586–591 (2018).6. J. Gu et al., Science 364, 1068–1075 (2019).7. Y. Feng et al., Nature 491, 478–482 (2012).8. Y. Yang et al., J. Med. Chem. 60, 1994–2005 (2017).

FIGURE 1. Structures of the cellular respiration–related protein complexes. (A) Overall structure of supercomplex (SC) I1III2IV1 viewed along the mitochondrial inner membrane. The surface models in different colors represent different subunits of the SC. (B) Overall structure of megacomplex I2III2IV2 viewed from the mitochondrial matrix. The surface models in different colors represent different subunits of the megacomplex. The sites representing the structural basis of the quinone pool are labeled. (C) Overall structure of adenosine triphosphate (ATP) synthase tetramer viewed from the mitochondrial matrix. The catalytic state of each monomer is indicated. Two inhibitory factor 1 (IF1) dimers linking within the tetramer are labeled. The surface models in different colors represent different subunits of ATP synthase. (D) ATP synthase dimer bending the cristae ridge. Each segment of ATP synthase is labeled. The cristae membrane is represented by a curved dash line. The surface models in different colors represent different subunits of ATP synthase. (E) Overall structure of Ndi1. Ndi1 is shown in ribbon model and colored rainbow. The bound NADH, flavin adenine dinucleotide (FAD), and UQ5 molecules are shown in stick model and labeled. (F) Overall structure of Plasmodium falciparum NDH2 (PfNDH2). Two monomers of PfNDH2 dimer are shown in orange and violet, respectively. The bound FAD and inhibitory compound 2 molecules are shown in stick model and labeled.

CHAPTER 1 13

The Research Group of Hang ShiHang Shi

Hang Shi received her undergraduate education from the Department of Physics at Tsinghua University from 1992 to 1996. After graduation, she moved to the United States and received graduate training in structural biology at the Cold Spring Harbor Laboratory. She obtained her doctorate from the Department of Physics and Astronomy at the University of Massachusetts, Amherst, in 2003. She then pursued postdoctoral training and later worked as a research scientist in the laboratories of James H. Hurley in the National Institute of Diabetes and Digestive and Kidney Diseases (U.S. National Institutes of Health) and Günter Blobel (1999 Nobel Laureate in Physiology or Medicine) at Rockefeller University between 2004 and 2017. A biophysicist by training, she has applied crystallography, cryo-electron microscopy, computation, and other technologies from different disciplines in her research and studied the different aspects of RNA biology as well as intracellular trafficking. In 2017, she returned to Tsinghua University and started her own research program.

Hang’s group consists of two graduate students, three undergraduate students, and several staff scientists. Her group continues to use multidisciplinary approaches to tackle problems in RNA biology. As the leader of a new group, her major focus is to develop a pipeline that enables the efficient determination of RNA structures to facilitate the understanding and application of RNA in both biological and medical research.

Since biology entered its molecular era, which was marked by the first determination of a crystal structure (β-globin) in 1960, RNA structure determinations and the mechanism of RNA folding have continued to be major challenges in the fields of both RNA and structural biology. After analyzing RNA folding using computational tools, Hang speculated that although the intrinsic flexibility of RNA was the major cause of failures of RNA structure determinations, well-folded stable RNAs should exist, similar to well-known examples such as ribosomes, among the vast number of noncoding RNAs. However, the routine techniques used in biomedical research have been unable to measure RNA stability quantitatively, thus preventing their efficient identification. She believed that if such methods could be established, along with improved sample preparation and structure stabilization, then a structural genomics approach could be adopted to improve our knowledge of RNA folding.

Hang began to apply force spectroscopy techniques in 2017 to quantitatively analyze RNA structure and stability (Figure 1). Previously, such techniques were used primarily by single-molecule biologists. She first selected RNA based on computational analysis and then used acoustic force spectroscopy and optical tweezers to compare differences in their free energy change upon folding. In addition, she used these technologies to investigate the effects of

chemical modifications on RNA folding. Because these techniques had not been adapted for biologists, she developed the first automated data analysis software for force spectroscopy to improve throughput and user experience. Meanwhile, she also worked on new chemical tools and methods that can reduce the flexibility of RNA structure. She continued to develop hardware, software, and experimental procedures to establish tools and methods for high-throughput force spectroscopy screening and structure determination.

Hang is also searching for new regulatory signals on RNA and working to understand the effects of RNA editing from a structural point of view. Her research is funded by the Beijing Advanced Innovation Center for Structural Biology, National Natural Science Foundation of China, and Tsinghua University. Hang’s long-term goal is to understand RNA folding and how it connects to its biological functions. She is also working to make the tools and methods developed in her laboratory available to the biology community.

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,��Tsinghua�University,�Beijing,�[email protected]

FIGURE 1. Flow chart of the multidisciplinary approach for RNA structure determination (unpublished data).


FIGURE 1. Structure and mechanogating mechanism of the Piezo channel. (A) The propeller-shaped cryo-electron microscopy structure of Piezo2. (B) The three highly curved transmembrane blades, marked by the green dashed lines, form a nanobowl shape with the indicated depth, open diameter, surface area, and projection area. (C) and (D) illustrate the 38-transmembrane (TM) topology comprised of nine repetitive transmembrane helical units (THUs) of four transmembrane helices each, the outer helix (OH), the pore-lining inner helix (IH), and other featured domains such as Beam, Cap, and Anchor. (E) The central pore module. (F) Structural comparison of the central pore of Piezo1 and Piezo2. (G) Radius of the vertical central pore of Piezo1 and Piezo2. Notably, the TM gate is in a closed state in Piezo2, but in an open state in Piezo1, while the putative cytosolic gate (the constriction neck) remains closed in both Piezo1 and Piezo2. (H) Mutants without the cap domain cannot mediate mechanically activated currents. The figure was adapted from (4).

The Research Group of Bailong XiaoBailong Xiao

Bailong Xiao obtained his bachelor ’s degree from the Department of Biochemistry at Sun Yat-sen University in 2001. He then pursued his doctoral studies with Wayne Chen from the University of Calgary in Canada from 2001 to 2006, focusing on the cardiac ryanodine receptor. Subsequently, he undertook his postdoctoral training with Ardem Patapoutian at the Scripps Research Institute in the United States from 2007 to 2012. There, he characterized several classes of ion channels involved in sensing noxious chemicals, temperature, and mechanical force (1). In 2013, he was appointed as a tenure-track assistant professor and was then promoted as a tenured associate professor in 2018 at the School of Pharmaceutical Sciences in Tsinghua University. To date, Xiao has trained six Ph.D. graduate students and one postdoctoral fellow. His research group currently consists of two postdoctoral fellows and 13 Ph.D. graduate students.

Xiao’s team works on Piezo1 and Piezo2, which are mechanically activated cation channels that determine various mechanotransduc-tion processes, including the sense of touch, proprioception, and tactile pain, and regulate vascular development, blood pressure, and bone formation. Piezo channels also serve as validated therapeutic targets. Taking a multidisciplinary approach, Xiao’s research group is working to understand how Piezo channels effectively convert mechanical force into electrochemical signals, with the ultimate goal

of developing novel therapeutics and technologies. Toward these ends, Xiao’s research group has determined the medium- and high-resolution cryo-electron microscopy structures of the full-length 2,547-residue mouse Piezo1 (2, 3), and then the high-resolution structure of the full-length 2,822-residue mouse Piezo2, with a complete 38-transmembrane–helix topology (4).

The Piezo channel forms a homotrimeric structure that resembles a three-bladed propeller. It comprises three highly curved transmembrane blades and a centrally embedded extracellular cap (Figure 1 A–D). On the basis of structural and functional analyses (3–8), Xiao’s group proposed that the Piezo channel might employ the top cap to control the transmembrane gate, while the blades function as a lever-like apparatus to allosterically control the putative cytosolic gate. Furthermore, they have revealed the physiological role of Piezo2 in suppressing acute pain (9), while Piezo1 mediates mechanical load-dependent bone formation (10).

References1. B. Coste et al., Nature 483, 176–181 (2012).2. J. Ge et al., Nature 527, 64–69 (2015).3. Q. Zhao et al., Nature 554, 487–492 (2018).4. L. Wang et al., Nature 573, 225–229 (2019).5. Q. Zhao et al., Neuron 89, 1248–1263 (2016).6. T. Zhang et al., Nature Commun. 8, 1797 (2017).7. Y. Wang et al., Nature Commun. 9, 1300 (2018).8. B. Xiao, Ann. Rev. Pharmacol. Toxicol. 60, 1–24 (2019). 9. M. Zhang et al., Cell Rep. 26, 1419–1431 e1414 (2019).10. W. Sun et al., eLife 8, e47454 (2019).


CHAPTER 1 15

The Research Group of Haipeng GongHaipeng Gong

Haipeng Gong received his bachelor ’s and Master ’s degrees from the Department of Biological Sciences and Technologies at Tsinghua University in 1997 and 2000, respectively. He pursued his doctoral training in computational studies of protein structures in the laboratory of George Rose and received his Ph.D. from the Department of Biophysics at the Johns Hopkins University in 2006. After graduation, Gong continued his work on protein folding/unfolding simulations in the same laboratory as a postdoctoral fellow for one year. In 2007, he moved to the laboratory of Tobin Sosnick as part of the Program of Biochemistry and Molecular Biology at the University of Chicago as a postdoctoral fellow, where he was involved in algorithm development for protein structure prediction and refinement. In 2009, Gong joined the School of Life Sciences at the Tsinghua University as an assistant professor. He was promoted to associate professor in 2015.

Gong’s group currently consists of six graduate students, who are studying the structures and molecular mechanisms of biological macromolecules, principally proteins, using existing or newly developed computational approaches. Specifically, their research focuses on two main areas: predicting protein structure from amino acid sequences and investigating protein dynamics using molecular dynamics simulations. The Gong group has developed a series of computer algorithms for various protein-structure prediction tasks and has published more than 30 scientific articles in peer-reviewed journals, including Nature Machine Intelligence, PLOS Computational Biology, Bioinformatics, and Journal of Physical Chemistry Letters. The main contributions of Gong’s group are summarized below.

1. Development of methods for protein structure predictionThe three-dimensional structure of a protein is essential for its

proper functioning, but it is impractical to experimentally determine the structures of hundreds of millions of proteins. Being able to predict protein structures from their amino acid sequences is therefore of great importance. Traditional de novo protein structure

prediction methods rely on intensive computer simulations to locate the lowest energy structure by sampling the conformational space, and thus their success is limited by the accuracy of potential energy estimation and the efficiency of conformational sampling algorithms. Gong’s group has designed a well-performing statistical potential ORDER_AVE (1) for accurate evaluation of protein structures, and developed the machine-learning-based algorithms LRFragLib (2) and DeepFragLib (3) to improve the quality of selected fragments, and thus the sampling efficiency, for fragment-assembly-based folding simulations.

Recently, the prediction of residue contacts has become a more attractive approach, because the predicted contact map can assist the structure modeling of proteins. Gong’s group has developed several algorithms to predict residue contacts [e.g., DeepConPred2 (4) and AmoebaContact (5)] and to refine the predicted residue contact maps [e.g., RDb2C (6)]. Moreover, they have designed a gradient descent-based algorithm, GDFold (5), to quickly model protein structures using predicted residue contacts.

2. Investigation of molecular mechanisms using computer simulations

Translocation of matter across the membrane bilayer requires membrane proteins like transporters and channels. Both direct translocation and its regulation require large-scale conformational changes of membrane transporters and channels. Gong’s group adopts molecular dynamics simulations to observe and quantify these conformational changes and has published a series of papers exploring the mechanisms of ion selection and voltage activation in a number of voltage-gated sodium channels (7, 8). Additionally, the group has investigated the cycle of conformational changes for various membrane transporters (9).

References1. Y. Liu, J. Zeng, H. Gong, Proteins 82, 2383–2393 (2014).2. T. Wang et al., Bioinformatics 33, 677–684 (2016).3. T. Wang et al., Nat. Mach. Intell. 1, 347–355 (2019).4. W. Ding et al., Comput. Struct. Biotechnol. J. 16, 503–510 (2018).5. W. Mao, W. Ding, H. Gong, ArXiv, arXiv:1905.11640 (2019).6. W. Mao et al., BMC Bioinformatics 19, 146 (2018).7. J. Zhang et al., Protein Cell 9, 580–585 (2018).8. R. N. Sun, H. Gong, J. Phys. Chem. Lett. 8, 901–908 (2017).9. M. Ke et al., PLOS Comput. Biol. 13, e1005603 (2017).Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�

Tsinghua�University,�Beijing,�[email protected]


The Research Group of Hong-Wei WangYuanyuan Fan

Hong-Wei Wang received his bachelor ’s degree in biological sciences and biotechnology from Tsinghua University in 1996, where he subsequently earned his doctorate degree under the supervision of Sen-Fang Sui in 2001. He then worked as a postdoctoral fellow under the supervision of Eva Nogales, advancing to the position of research scientist in 2006 at Lawrence Berkeley National Laboratory in Berkeley, California. He joined Yale University as a tenure-track assistant professor of molecular biophysics and biochemistry in 2009 and returned to Tsinghua University as a professor of life sciences in December, 2010, where he is currently dean of the School of Life Sciences.

Wang holds numerous awards, including a prize from the National Science Fund for Distinguished Young Scholars (2018), the 2nd Chinese Cryo-EM Outstanding Contribution Award (2019), the 11th C. C. Tan Life Science Innovation Award (2018), the 2018 Beijing Teachers’ Role Model Award, and the 2017 Beijing Outstanding Teacher Award. He currently serves as an editorial board member of the Journal of Biological Chemistry and Biophysics Reports and as an executive editor of Biochemistry. He was also elected chair of the 2019 Gordon Research Conference on Three-Dimensional Electron Microscopy and is currently the chair of the Cryo-Electron Microscopy Subsociety of the Biophysical Society of China and a council member of the Biophysical Society of China, the Chinese Electron Microscopy Society, and China Instrument and Control Society.

Wang’s research group currently consists of three postdoctoral fellows, three staff members, and 15 graduate students. The group’s research focuses on the development and application of cryo-electron microscopy (cryo-EM) and determining the structures and mechanisms of macromolecular complexes for

nucleic acid quality control. To date, the group has published more than 90 peer-reviewed articles in prestigious journals, including Science, Cell, Nature, Nature Structural & Molecular Biology, Nature Communications, Journal of the American Chemical Society, Cell Research, and Structure. The major contributions of Wang’s team are as follows.

1. Development of novel imaging methods to obtain atomic-resolution structures of macromolecules

The Volta phase plate (VPP) has been developed as a new generation of phase plate devices for transmission electron microscopy (TEM). The VPP substantially boosts the image contrast of biological cryo-EM samples, enabling the near-atomic resolution structural determination of proteins with low molecular weights (1–3). Using a simulation approach, Wang’s group obtained evidence for a novel theory that overfocus and underfocus imaging become indistinguishable when applying the VPP on objective-lens spherical aberration (Cs)-corrected cryo-EM (4). The structure of human apoferritin at ~3-Å resolution in over- and underfocus conditions was determined using a VPP-Cs-corrector-coupled TEM system, supporting the practicability and efficiency of the new imaging strategy. These results not only revealed interesting properties of VPP-Cs-corrector-coupled TEM but also demonstrated the feasibility and advantages of such a system for high-resolution cryo-EM.

2. Development of novel supporting grids for high-resolution cryo-EM-based structural determination

Cryo-EM is now an important tool in structural biology research. However, cryo-specimen preparation remains a major challenge. The absorption of macromolecules at the air–water interface is frequently observed when using canonical supporting films in cryo-EM, resulting in partial denaturation and/or preferential orientations (5–6). Wang’s group recently developed affinity-ligand functionalized single-crystalline monolayer graphene membranes as novel cryo-EM supporting films (7). The functionalized graphene membrane has high conductivity and mechanical strength when irradiated as observed with EM, as well as high affinity and selectivity

FIGURE 2. Schematic diagram of the functionalized monolayer graphene membrane for high-resolution cryo-electron microscopy.


FIGURE 3. Single-particle analysis of the streptavidin specimen.

FIGURE 1. Image formation in overfocus and underfocus conditions. Micrographs collected in both conditions could contribute to near-atomic resolution reconstruction.

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for target biomolecules. These properties effectively prevent biomolecules from contacting the air–water interface and enable the reconstruction of macromolecules at near-atomic resolution by cryo-EM. The functionalized graphene membrane grids are expected to improve the reproducibility and robustness of cryo-EM specimens, thereby increasing the efficiency and throughput of high-resolution cryo-EM structural determination.

3. Establishing a new record for the structural determination of low-molecular-weight proteins by single-particle cryo-EM at near-atomic resolution

Owing to recent technical breakthroughs, single-particle cryo-EM has become a powerful tool for determination of near-atomic resolution structures of biochemically well-behaved “large” proteins (i.e., >300 kDa) (8). However, the low contrast of low-molecular-weight samples (i.e., <100 kDa) in cryo-EM imaging limits high-resolution structural analyses. Wang’s group applied their previously developed VPP-Cs-corrector-coupled cryo-EM imaging strategy and graphene supporting film for cryosample preparation to study streptavidin (SA), which has a molecular weight of only 52 kDa (4, 9). They successfully revealed two states of SA, with and without biotin-binding, at ~3-Å resolution by cryo-EM. Detailed image processing and analyses further suggested that the method could be applied to molecules as small as ~40 kDa. Of note, they also found that avoiding the adsorption of proteins at the air–water interface is critical for solving the high-resolution structure of target proteins (10).

4. Solving the high-resolution structures of human Dicer and its complex with RNA substrates

Human Dicer (hDicer), a member of the RNase III family, is a 220-kDa endonuclease consisting of multiple domains (11). Dicer is a key factor in the processing of double-stranded RNA (dsRNA) substrates into products of approximately 21–25 nucleotides in the RNA interference (RNAi) pathway (12, 13). After many years of effort, Wang and colleagues first reported a 4.4-Å resolution cryo-EM structure of hDicer bound to transactivation response RNA binding protein (TRBP), a dsRNA-binding protein with important roles in the regulation of dicing fidelity. The unusual L-shaped domain arrangement could explain the mechanism underlying Dicer activity, especially RNA cleavage-site selection. They reconstituted the hDicer-TRBP in complex in vitro with pre-let-7 RNA, a classic substrate of hDicer. Cryo-EM showed two distinct conformations of pre-let-7 bound to the hDicer-TRBP complex, one with a perfectly matched stem and another in which the stem splays near its loop.

An RNase A digestion assay further suggested that the hDicer-TRBP complex can stabilize substrates by annealing to their stems. This might explain why hDicer can recognize thousands of pre-microRNA substrates with different secondary structures and process these RNAs into similar products (14).

5. Solving the structures of ZAR1, RKS1, and PBL2 with different statuses by cryo-EM

An important mechanism by which both plants and animals defend against pathogens involves cytoplasmic receptors called NOD-like receptors (NLRs), which recognize molecules secreted by microorganisms that invade plant cells. Plant cell recognition of effectors results in cell death, thereby confining microbes to the site of infection. To determine the detailed mechanisms underlying the functions of plant NLRs, Wang’s group worked collaboratively with the groups of Jijie Chai and Jianmin Zhou to evaluate three proteins: ZAR1 (a highly conserved plant protein with broad functional importance via interactions with multiple “guardees” to recognize bacterial effectors), RKS1, and PBL2 (15). Using structural modeling by cryo-EM, they found that after infection, the bacterial effector modifies the plant “guardee” PBL2. This activates RKS1, resulting in substantial conformational changes, allowing plants to swap adenosine diphosphate (ADP) for adenosine triphosphate (ATP) and resulting in the assembly of a pentameric, wheel-like structure that was termed the “ZAR1 resistosome” (16, 17).

6. Solving the near-atomic resolution cryo-EM structure of the multisubunit tethering exocyst complex

In eukaryotic cells, the orderly and accurate transport, docking, tethering, and fusion of intracellular vesicles to target membranes form an elaborate vesicular trafficking network (18, 19). A detailed description of the structure and assembly of multisubunit tethering

FIGURE 4. Model of pre-microRNA recognition and processing by the hDicer–TRBP complex .

FIGURE 5. Molecular mechanisms contributing to the plant resistosome. Coiled-coil (CC), nucleotide-binding domain (NBD), helical domain (HD1), winged-helix domain (WHD), leucine-rich repeat (LRR), resistance-related kinase 1 (RKS1, a pseudokinase belonging to receptor-like cytoplasmic kinase subfamily XII-2), probable serine/threonine-protein kinase (PBL2), plasma membrane (PM).


complexes (MTCs) that initially tether vesicles and their target membranes is essential for understanding these processes. By incorporating cryo-EM and chemical cross-linking mass spectrometry (CXMS), Wang’s group, in collaboration with the groups of Wei Guo and Meng-Qiu Dong, solved the first intact Saccharomyces cerevisiae exocyst complex structure, which

functions in the tethering of secretory vesicles to the budding plasma membrane, at an average resolution of approximately 4.4 Å. The model provides insights into the hierarchical assembly of the exocyst complex, mainly mediated by assembly of the newly defined CorEx motifs. Additional cell biological data indicated that the Sec3 CorEx motif is essential for the recruitment of the other seven exocyst subunits and the tethering of secretory vesicles. This work provides a basis for understanding the assembly and function of other MTCs and will ultimately clarify the spatiotemporal molecular events in vesicular trafficking (20).

Technological and methodological advances in high-resolution cryo-EM are important areas of future research by Wang and colleagues. His group will continue their work in the development of new cryo-EM specimen preparation methods and imaging strategies. They aim to apply these new methods to studies of nucleic acid quality control, especially analysis of the exosome complex with its various cofactors, hDicer and RNA-induced silencing complex (RISC)-loading complexes in RNAi pathways, long noncoding RNAs in complex with their protein partners, and DNA repair complexes with their DNA substrates. They will also collaborate with other scientists to decipher the mechanisms underlying the functions of important molecular machines by cryo-EM.

References1. R. Danev et al., Proc. Natl. Acad. Sci. U. S. A. 111, 15635–15640 (2014).2. R. Danev et al., eLife 5, e13046 (2016).3. D. Tegunov et al., eLife 6, e23006 (2017).4. X. Fan et al., Structure 25, 1623–1630 (2017).5. R. M. Glaeser et al., Curr. Opin. Colloid Interface Sci. 34, 1–8 (2018).6. R. M. Glaeser et al., Biophys. Rep. 3, 1–7 (2017).7. N. Liu et al., J. Am. Chem. Soc. 141, 4016–4025 (2019).8. M. Liao et al., Nature 504, 113–118 (2013).9. J. Zhang et al., Adv. Mater. 29, 1700639 (2017).10. X. Fan et al., Nat. Commun. 10, 2386 (2019).11. H. Zhang et al., Cell 118, 57–68 (2004).12. E. Bernstein et al., Nature 409, 363–366 (2001).13. S. M. Elbashir et al., Genes. Dev. 15, 188–200 (2001).14. Z. Liu et al., Cell 173, 1191–1203 (2018).15. J. L. Dangl et al., Science 364, 31–32 (2019).16. J. Wang et al., Science 364, 43 (2019).17. J. Wang et al., Science 364, 44 (2019).18. I. M. Yu et al., Dev. Biol. 26, 137–156 (2010).19. S. R. Pfeer et al., Nat. Cell. Biol. 1, E17–E22 (1999).20. K. R. Mei et al., Nat. Struct. Mol. Biol. 15, 6483–6494 (2018).

The Research Group of Xueming Li Xueming Li

Xueming Li studied electron microscopy in condensed matter physics and received his doctorate from the Institute of Physics at the Chinese Academy of Sciences (Beijing, China) in 2009. He then joined the laboratory of Yifan Cheng at the University of California, San Francisco, as a postdoctoral scholar and changed his research direction to focus on cryo-electron microscopy (cryo-EM) in

structural biology. Between 2009 and 2014, he developed methods that realized atomic-resolution cryo-EM.He is one of the earliest researchers to have integrated a graphics processing unit (GPU) into the cryo-EM field (1). His work on electron counting cameras and motion correction (2, 3) greatly contributed to the “resolution revolution” that has occurred in cryo-EM since 2013. In 2014, Li returned to China and was appointed a principal investigator at Tsinghua University.

Li is currently a tenured associate professor of the School of Life Sciences and a principal investigator of the Beijing Advanced Innovation Center for Structural Biology and Tsinghua-Peking Joint Center for Life Sciences. In 2015, he was selected by the Thousand Young Talents Program, State Council of China and received the Qiu Shi Outstanding Young Scholar Award. He received the National Science Fund for Distinguished Young Scholars in 2017.

Li’s research group currently has more than 20 members,

FIGURE 6. Hierarchical assembly of exocyst structure. (A) Composition and interactions between two exocyst subcomplex subunits. (B) Two subunits assemble via the CorEx motifs. (C) Two subunits are packed into subcomplex I and subcomplex II with the CorEx motif to form a four-helix bundle. (D) Subcomplex I and subcomplex II finally assemble into the holo-exocyst complex.

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�Tsinghua-Peking�Joint�Center�for�Life�Sciences,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�[email protected]

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including graduate students, postdoctoral fellows, and technicians. His group’s interests include the structures and functions of biomacromolecules under physiological conditions and the development of experimental and computational methods to push the resolution and general applicability of cryo-EM. Thus far, Li’s group has published more than 20 scientific articles in high-impact journals, including Nature and its branch journals. The major contributions made by Li’s research group are summarized below.

1. Using a particle f ilter algorithm to achieve robust and efficient cryo-EM 3D reconstruction

As a tool for determining the atomic structures of biological macromolecules in solution, single-particle cryo-EM reconstructs 3D-density maps by estimating orientation parameters from a series of input images of randomly oriented macromolecules. Li’s team adopted a particle filter algorithm (4) that describes the parameter estimation problem as a posterior probability density function of the parameter estimates (Figure 1). They established a general Bayesian framework based on the particle filter and implemented it in a program named THUNDER (available through http://github.com/THUEM/thunder), which features self-adaptive parameter optimization, tolerance to bad particles, and per-particle defocus refinement.

2. Removing bottlenecks in electron crystallography of microcrystals

The electron diffraction method for three-dimensional (3D) microcrystals, termed MicroED, is now a popular technology for determining the structures of small crystals of proteins or other small molecules. Li’s team developed a stage-camera synchronization scheme and implemented it in a data-acquisition program called eTasED (available through http://github.com/THUEM/eTasED) that enables MicroED on a conventional cryo-electron microscope. without a movie-mode camera (5). Preparing a thin crystal sample for cryo-EM, typically no thicker than 0.5 μm, is always a challenge. By applying a focused ion beam (FIB) during

sample preparation in MicroED and studying the influence of FIB milling, Li’s group developed a reliable method for preparing high-quality MicroED specimens (6), and combined the two techniques mentioned above to solve a series of ultra-high-resolution structures up to 0.6-Å resolution with visible hydrogen atoms (Figure 2).

3. Introducing deep-learning technology to single-particle cryo-EM

Selecting protein particles from cryo-EM micrographs is a time-consuming step but is essential for atomic-resolution 3D reconstruction. Li’s team developed the first deep-learning approach to solving this problem, a framework named DeepPicker, which trains a computer to recognize and pick particles using a convolution neural network (7). Deep learning is becoming a popular tool in cryo-EM and is increasingly applied not only to single-particle analysis, but also cryo-electron tomography (cryo-ET) technology.

4. Revealing the structural basis of bacterial secretion systemsBacteria have developed special secretion systems to secrete

a wide range of intracellular materials, including proteins and DNA, into the extracellular milieu or host cells. These systems are also responsible for secreting many vital protein toxins, thereby playing key roles in bacterial pathogenesis. Li’s group studied the large outer-membrane complexes of several secretion systems using cryo-EM, including a GspD secretin channel (8) and a secretin–pilotin GspD–AspS complex (9) in a Type II secretion system (Figure 3a), as well as a membrane core complex in a Type VI secretion system (Figure 3b) (10). These structures provided a basis for understanding the dynamic processes of secretion and corresponding complex assembly.

5. Applications on biological questionsLi’s group also collaborated with other laboratories to apply

their advanced cryo-EM technology to other biological questions, especially to protein complexes with large conformational

Round 4 (53 Å) Global

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FIGURE 1. Illustration of an orientation parameter estimation procedure (4). A cyclic nucleotide-gated channel data set was used for the illustration. Three-dimensional reconstructions (gray maps) and corresponding likelihood functions (color figures) of the estimates in different rounds of the reconstruction are shown.

0.65 Å

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0.59 Å

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v. Ultrahigh resolution diff ractions and solved structures by MicroED. (A) A typical diffraction pattern and (B) solved ultrahigh-resolution structure with visible hydrogen atoms (5).


The Research Group ofQiangfeng Cliff ZhangQiangfeng Cliff Zhang

Qiangfeng Cliff Zhang received his bachelor ’s and doctoral degrees in computer science from the University of Science and Technology of China in 2000 and 2006, respectively, working on algorithm design and analysis. He earned a second doctorate from the Department of Biochemistry and Molecular Biophysics at Columbia University in 2012, where he studied computational structural biology in the laboratory of Barry Honig. Zhang then moved to Stanford University and completed his postdoctoral training, focusing on analyzing the RNA secondary structure using sequencing approaches while jointly supervised by Howard Chang and Mike Snyder. In 2015, he joined Tsinghua University as an assistant professor and was promoted to associate professor in 2018.

Zhang’s current research group consists of one staff member, four postdoctoral fellows, and 15 graduate students. Working at the interface of structural biology, genomics, machine learning, and big data analysis, the primary goal of the group is to develop and apply novel techniques to enable the study of structural systems biology

and promote the field. The major contributions made by the group to date are summarized as follows.

1. Developing transcriptome-wide technologies to probe RNA secondary structures and interactions by sequencing

The limited knowledge of RNA structure has restricted our understanding of its crucial roles in RNA function and regulation. Zhang coinvented a novel method called in vivo click selective 2-hydroxyl acylation analyzed by primer extension (icSHAPE) (Figure 1A) to measure RNA structures in vivo on a transcriptome-wide level (i.e., RNA structurome) by combining the use of a new SHAPE reagent (NAI-N3) and RNA sequencing (1). He also codeveloped psoralen analysis of RNA interactions and structures (PARIS) (Figure 1B), which combines psoralen cross-linking and sequencing to characterize both the RNA structurome and interactome in vivo (2). These technologies, along with those developed by other groups, have opened the door to the in vivo study of the structural and interaction landscape of the entire transcriptome, and have revealed the importance of a previously obscure layer of gene-expression regulation based on RNA structure and interactions (3).

2. Elucidating the critical roles of the in vivo RNA structure in RNA function and regulation

Similar to protein structure, RNA structure is crucial to the functional and regulatory roles of RNA, including splicing, modification, translation, processing, and degradation. In an

flexibilities. Using MicroED, they solved multiple important structures, including those of the α-synuclein amyloid fibril (11), a eukaryotic cyclic nucleotide-gated channel (12), human endolysosomal TRPML3 channels (13), complexes of chromatin remodeler Snf2 and nucleosomes in different states (14, 15), the Piezo1 and 2 channels (16), and the FUS (fused in sarcoma) reversible amyloid fibril core (17).

The long-term goal of Li’s group is to develop experimental and computational methods that achieve atomic-resolution cryo-EM with high efficiency and general applicability, involving single-particle analysis, cryo-ET, and electron crystallography.

References1. X. Li, N. Grigorieff, Y. Cheng, J. Struct. Biol. 172, 407–412 (2010).2. X. Li et al., Nat. Methods 10, 584–590 (2013).

3. X. Li et al., J. Struct. Biol. 184, 251–260 (2013).4. M. Hu et al., Nat. Methods 15, 1083–1089 (2018).5. H. Zhou et al., Anal. Chem. 91, 10996–11003 (2019).6. H. Zhou, Z. Luo, X. Li, J. Struct. Biol. 205, 59–64 (2019).7. F. Wang et al., J. Struct. Biol. 195, 325–336 (2016).8. Z. Yan et al., Nat. Struct. Mol. Biol. 24, 177–183 (2017).9. M. Yin, Z. Yan, X. Li, Nat. Microbiol. 3, 581–587 (2018).10. M. Yin, Z. Yan, X. Li, Cell Res. 29, 251–253 (2019).11. Y. Li et al., Cell Res. 28, 897–903 (2018).12. M. Li et al., Nature 542, 60–65 (2017).13. X. Zhou et al., Nat. Struct. Mol. Biol. 24, 1146–1154 (2017).14. X. Liu et al., Nature 544, 440–445 (2017).15. M. Li et al., Nature 567, 409–413 (2019).16. Q. Zhao et al., Nature 554, 487–492 (2018).17. F. Luo et al., Nat. Struct. Mol. Biol. 25, 341–346 (2018).

FIGURE 3. Structures underlying bacterial secretion systems. (A) cryo-EM density maps of the pilotin–secretin AspS–GspD complex of T2SS and (B) the membrane core complex of T6SS (10).

OMOM

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21CHAPTER 2

FIGURE 1. Novel methods to explore RNA structures and interactions. (Left) icSHAPE uses NAI-N3, a shape reagent that penetrates the cell membrane and targets unstructured, flexible, single-stranded nucleotides, which are then detected by deep sequencing and bioinformatics analysis. (Right) PARIS uses a psoralen derivative, 4'-aminomethyl trioxsalen (AMT), to treat cells, followed by 2D gel separation to enrich cross-linked RNA duplexes, which are then submitted to proximity ligation and sequencing library construction, and finally identified by bioinformatics analysis.

FIGURE 2. Establishing RNA structural maps of two Zika virus (ZIKV) genomes in vivo by combining icSHAPE, PARIS, and evolutionary comparative analysis . Comparative analysis revealed both common and lineage-specific RNA structural elements, including a long-range interaction between the 5'UTR and the coding region that affects virus infectivity and only exists in the Asian lineage. Adapted from (4). Copyright 2018, with permission from Elsevier.

Lower translation efficiency

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FIGURE 3. Cytotopic RNA structure maps of chromatin, nuclear, and cytosolic subcellular compartments, combined with subsequent comparative studies, reveal the interplay between RNA structural changes, protein binding, and RNA modification as well as the central role of the RNA secondary structure in connecting transcription, translation, and RNA degradation.

FIGURE 4. A2-Net formulates the construction of molecular structural models from cryo-electron microscopy density volumes as a 3D detection problem and uses a deep-learning framework that includes amino acid determination and a sequence-guided Monte Carlo Tree Search to thread candidate amino acids into a molecular structure.


The Research Group of Sai Li Sai Li

Sai Li received his bachelor ’s degree from the School of Physics and Technology, Wuhan University, China, in 2006. He obtained his doctorate in biophysics from the Faculty of Physics, Georg-August University of Göttingen, Germany, in 2012. In late 2012, he started his career in cryo-electron tomography (cryo-ET), subtomogram averaging (STA), and structural virology at the Oxford Particle Imaging Centre, University of Oxford, United Kingdom, as a postdoctoral assistant, and was promoted to senior research associate in 2017. He returned to China in 2018 as a principal investigator at the School of Life Sciences, Tsinghua University, Beijing.

Li leads one of the youngest research groups at the Beijing Advanced Innovation Center for Structural Biology. His lab consists of a postdoctoral fellow, a lab manager, and three graduate students. The group focuses on cryo-ET and its applications for in situ structural biology, with the long-term goal of bridging dimensions between structures at subnanometer resolution and ultrastructural landscapes at the micrometer scale in native contexts. Some of his laboratory’s major contributions are described below.

1. Development and implementation of cryo-ET and STA methodsThe group is active in developing cryo-ET techniques, especially

in the direction of molecular tomography and subnanometer-resolution STA. By performing thorough comparisons between STA and single-particle analyses (SPA) to solve pleomorphic macromolecular complexes, the group aims to identify and overcome the limitations of STA in achieving near-atomic resolution. Recently, they made progress in breaking the 7-Å resolution barrier using customized STA methods (unpublished), which was better

than the highest resolution achieved by SPA on an identical sample. Efforts are being made to enable automatic and high-throughput tomographic data collection and processing at the Tsinghua cryo-EM facility, where the hope is to make high-resolution tomography routinely accessible.

Li’s vision also includes bridging dimensions between subnanometer-resolution structures to micrometer scale in in situ landscapes, a soaring demand in the field of structural biology. Workflows that include sets of cutting-edge techniques, such as focused-ion beam milling or correlative light and electron microscopy in cryogenic conditions, will be implemented to bridge molecular and cellular tomography. These solutions will provide unprecedented resolution for cell biology studies and will become a key module at the Beijing Advanced Innovation Center for Structural Biology. Progress toward reaching this goal can be seen in Li’s pioneering work: capturing a transient intermediate structure of virus–host membrane fusion (1), and solving the structure of a 57-kDa membrane protein at ~16-Å resolution from Volta phase-plate tomographic data (2).

2. Native structures and cellular entry of emerging enveloped viruses

Since 2009, Li has been working on gaining insights into the native structures and cellular entry of emerging enveloped viruses. The enveloped viruses he has studied include the influenza virus (3, 4), Lassa virus (5, 6), hantavirus (7, 8), phlebovirus (1), and pleolipovirus (2). In the future, the group will combine multiple biochemical and structural techniques to study viral structures and virus-host interactions. References

1. S. Halldorsson et al., Nat. Commun. 9, 349 (2018).2. K. El Omari et al., Nat. Commun. 10, 846 (2019).3. S. Li et al., Biophys. J. 106, 1447–1456 (2014).4. S. Li et al., Biophys. J. 100, 637–645 (2011).5. Y. Watanabe et al., Proc. Natl. Acad. Sci. U. S. A. 115, 7320–7325 (2018).6. S. Li et al., PLOS Pathog. 12, e1005418 (2016).7. I. Rissanen et al., J. Virol. 91 (2017).8. S. Li et al., Cell Rep. 15, 959–967 (2016).

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�ChinaCorresponding�author:�[email protected]

interesting application of RNA structure probing, Zhang’s group determined the RNA genome structure of the Zika virus (ZIKV) in infected cells. As an RNA virus, the life cycle of ZIKV is tightly regulated at the RNA level (4). The study showed, for the first time, that RNA structures and interactions in the coding sequence region are important for viral infection, resulting in the proposal of RNA structures as novel targets for drug discovery (Figure 2).

In another study, the group resolved comprehensive RNA structure maps for both human and mouse cell lines in subcellular compartments using icSHAPE (5). The cytotopic RNA structure maps revealed a central role of RNA structure in biological processes. The group also discovered the interplay among RNA structural changes, RNA-binding proteins (RBPs), and chemical modifications. This approach proved to be especially valuable for gaining insight into epitranscriptomic RNA chemical modification (Figure 3).

3. Developing computational algorithms and resources for structural systems biology

Another research focus of Zhang’s group is to develop computational techniques to enable studies in structural systems biology. Toward this end, the group developed an artificial intelligence method named prePPI, to model and predict protein–

protein interactions (6, 7). By combining machine-learning and coarse-grained structural modeling approaches using remote geometric relationships, PrePPI can predict protein–protein interactions on a genome-wide scale with quality comparable to that of high-throughput experiments. The group recently developed a new deep-learning method, A2-Net, to construct atomic structure models from cryo-electron microscopy density maps (Figure 4). This work represents an important step toward automatic structure model building (8).

References1. R. C. Spitale et al., Nature 519, 486–490 (2015).2. Z. Lu et al., Cell 165, 1267–1279 (2016).3. X. Qian, J. Zhao, P. Y. Yeung, Q. C. Zhang, C. K. Kwok, Trends Biochem. Sci. 44, 33–52

(2019).4. P. Li et al., Cell Host Microbe 24, 875–886 e875 (2018).5. L. Sun et al., Nat. Struct. Mol. Biol. 26, 322–330 (2019).6. Q. C. Zhang, D. Petrey, R. Norel, B. H. Honig, Proc. Natl. Acad. Sci. U. S. A. 107,

10896–10901 (2010).7. Q. C. Zhang et al., Nature 490, 556–560 (2012).8. K. Xu, Z. Wang, J. Shi, H. Li, Q. C. Zhang, in Proceedings of the AAAI Conference on

Artificial Intelligence (AAAI-19), vol. 33, no. 1, Hilton Hawaiian Village, Honolulu, Hawaii, USA, January 27–February 1, 2019 (AAAI Press, Palo Alto, CA), pp. 1230–1327.

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The Research Group of Yi Xue Yi Xue

Yi Xue received his bachelor ’s degree from the Department of Modern Applied Physics in 1997 and his Master 's degree from the Department of Biological Science and Biotechnology in 2003, both at Tsinghua University. In 2009, he received his doctorate from the Department of Chemistry at Purdue University, where he began a research career in the study of protein dynamics, using nuclear magnetic resonance (NMR) and computational tools. After working as a postdoctoral associate in the same laboratory for three years, he continued his postdoctoral training in Hashim Al-Hashimi’s laboratory at the University of Michigan, and later at Duke University, where the focus of his research changed to noncoding RNAs (ncRNAs). In 2016, he was designated as the principal investigator of the Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, and the Beijing Advanced Innovation Center for Structural Biology at Tsinghua University. Xue’s research group currently consists of 11 Ph.D. students, one technician, and one laboratory manager.

NMR spectroscopy is a powerful tool for solving three-dimensional (3D) structures of biological macromolecules. Other methods include X-ray crystallography and cryogenic electron microscopy, which can be viewed as “what you see is what you get” techniques, and are suitable for macromolecules with well-defined structures. In contrast, NMR adopts an indirect way to determine structures by combining experimental structural restraints and computational techniques, such as molecular dynamics simulations. Thus, NMR has unique capabilities in dealing with biological systems with prominent dynamics. The overarching research interests of Xue’s group are to develop and combine novel solution NMR techniques, labeling methods, and computational tools to characterize the conformational dynamics of ncRNAs, disordered proteins, and ribonucleoproteins. The main ongoing research projects of Xue’s research group are as follows:

1. Developing chemical shift prediction tools for nucleic acidsThe NMR chemical shift reflects subtle changes in molecular

conformation and is the most important NMR observable. Establishing an accurate relationship between the chemical shift and structure is often key to interpreting NMR data and constructing structural models. Relevant algorithms have been relatively well-developed for proteins, but for nucleic acids, such methodology remains in its infancy. Xue’s laboratory employs methods at different levels (empirical, semiempirical, and quantum mechanics/molecular mechanics) to predict chemical shifts from secondary or 3D RNA and DNA structures.

2. Characterizing the structural and dynamic aspects of long noncoding RNAs (lncRNAs) by NMR and computational approaches

In humans, protein-coding sequences account for less than 2% of the genome, while at least 60%–70% of total nucleotides are transcribed as ncRNAs, among which, most are lncRNAs. Although ncRNAs have been increasingly found to be critically important in many biological processes involving gene regulation and enzymatic catalysis, the structures and functions of the majority of ncRNAs remain unknown, presenting a significant challenge to biological scientists. The structural characterization of lncRNAs is particularly difficult because of their high flexibility and large size. Xue’s group aims to attack this challenging problem by combining recently developed RNA position-selective labeling schemes, novel chemical shift predictors, and molecular dynamics simulations, as well as an array of NMR methods such as spin relaxation, residual dipolar couplings, paramagnetic relaxation enhancement, pseudocontact shifts, and relaxation dispersion.

3. Reconstructing protein dynamics using X-ray diffraction data and molecular dynamics simulations

Protein functions are dictvated not only by structure, but also by conformational dynamics. Similar to the situation in solution, proteins in a crystalline environment undergo conformational dynamics over a broad timescale. The dynamics information of a protein crystal cannot be extracted satisfactorily using the current crystallography methods. Xue’s group aims at reconstructing protein dynamics using molecular dynamics simulations of a protein crystal restrained by experimental structure factors.



The research group of Xinquan WangXinquan Wang

Xinquan Wang received his bachelor ’s degree from the School of Life Sciences at Fudan University in 1995 and his doctorate from the Institute of Biophysics (IBP) at the Chinese Academy of Sciences (CAS) in 2000 under the supervision of Dongcai Liang and Wenrui Chang. After working in the IBP CAS for two years, he joined Christopher K. Garcia’s laboratory at Stanford University in 2003 for his postdoctoral research, in which he studied the structural mechanisms underlying the interactions of cytokines with their receptors. In 2008, Wang joined the Department of Biological Sciences and Biotechnology at Tsinghua University as a principal investigator. He was promoted to tenure-track associate professor in 2010 and to tenured professor in 2013. Currently, he is vice dean of the School of Life Sciences at Tsinghua University and vice director of the Beijing Advanced Innovation Center for Structural Biology.

Wang’s group is interested in the structural biology of cell surface receptors. One of the research targets of this group is the interleukin 1 (IL-1) family of cytokine receptors that plays critical roles in inflammatory response and regulation by recognizing IL-1 cytokines and activating cytokine signaling pathways. The research team aims to elucidate the macromolecular interactions mediating ligand recognition and activation of the IL-1 receptors. The second pursuit of the group involves cellular receptors of the highly pathogenic coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Wang’s group aims to understand the structural basis of virus-receptor binding and membrane fusion during infection by MERS-CoV and SARS-CoV. They are also interested in studying the epitopes and mechanisms of neutralizing monoclonal antibodies against MERS-CoV. Since 2008, Wang’s team has published more than 40 scientific articles in prestigious journals, including Nature Immunology, Science Translational Medicine, Nature Communications, PNAS, and Cell Research. The major scientific contributions made by Wang’s group are summarized below.

1. Elucidation of IL-1 cytokine recognition and assembly with IL-1 receptors

The IL-1 family of cytokines, consisting of 11 agonistic, antagonistic, or anti-inflammatory members, plays critical roles in inflammatory response and regulation during the onset and development of host reactions to different microbial and environmental challenges. Corresponding to the diverse effects of ligands, the IL-1 receptors include primary receptors for ligand-binding, shared coreceptors essential for signal transduction, and decoy and inhibitory receptors for negative regulation. Wang’s group had reported the crystal structure of IL-1β, in complex with IL-1RII and IL-1RAcP, which was the first ternary cytokine-receptor complex structure to be elucidated in the family, and provided a structural model for studying other IL-1 cytokines (Figure 1) (1). They also revealed specific interactions of IL-33 and IL-18 with their respective ligand-binding primary receptors ST2 and IL-18Rα by determining the corresponding complex structures (Figure 1) (2, 3). Besides studying static structural images, Wang’s group also systematically studied the behavior of IL-1 receptors in solution, using small-angle X-ray scattering, and discovered the functional relevance of the interdomain flexibility of IL-1 receptor for cytokine binding and signaling (Figure 1) (3, 4). Collectively, their work has

revealed the molecular details and dynamic nature of IL-1 receptors in recognizing cytokines and forming cytokine-receptor complexes capable of signal transduction.

2. Characterization of the structural features of MERS-CoV and SARS-CoV spike glycoprotein

In the 21st century, we have witnessed the outbreak of highly pathogenic SARS-CoV (2003) and MERS-CoV (2012), which caused respiratory infection with high case-fatality rates. These two human coronaviruses utilize the spike (S) glycoprotein (with subunits S1 and S2) on the envelope to recognize host cellular receptors and mediate the virus-cell membrane fusion through pre- to postfusion conformational changes. In collaboration with Ye Xiang and Linqi Zhang from the School of Medicine at Tsinghua University, Wang’s team characterized the structural features of SARS-CoV and MERS-CoV S glycoprotein in prefusion, receptor-bound, and postfusion states during viral entry, utilizing a combination of X-ray diffraction, cryo-electron microscopy (cryo-EM), and biochemical and virology studies (Figure 2). Shortly after the identification of MERS-CoV, Wang’s group revealed the molecular basis of its binding to host cell by determining the crystal structure of the receptor-binding domain (RBD) of MERS-CoV S glycoprotein in complex with the cellular receptor DPP4 (5). They also reported distinct conformations of the SARS-CoV S glycoprotein trimer in the prefusion state, resulting from the positional change of the RBD (6). These conformations have been shown to correspond to the inactive and active states of trimeric S for receptor binding; this state transition is required not only for receptor binding but also for antibody neutralization. Wang’s group further studied the cryo-EM structures of trimeric SARS-CoV S glycoprotein in complex with receptor ACE2 under various in vitro conditions that may mimic distinct conformations of the S glycoprotein during viral entry (7).

3. Revealing the neutralizing epitope and mechanism of antibodies targeting MERS-CoV

Isolation and characterization of neutralizing monoclonal antibodies (mAbs) is important for understanding the host protective response and developing therapeutic agents, such as vaccines, against viral infection. In collaboration with Linqi Zhang from the School of Medicine at Tsinghua University and Wenjie Tan from the Chinese Center for Disease Control, Wang’s team structurally characterized the epitopes and mechanisms of the potent mAbs MERS-4, MERS-27, MERS-GD27, and 7D10 (Figure 3) (8–12). Their studies revealed that MERS-4, MERS-27, and MERS-GD27 have distinct epitopes within MERS-CoV S RBD, representing the three different, currently known groups of RBD-

FIGURE 1. Recognition and assembly of IL-1 cytokines with their receptors .


25CHAPTER 3

targeting MERS-CoV antibodies. All three antibodies neutralize MERS-CoV infection by inhibiting the binding of S glycoprotein to its cellular receptor DPP4. Wang’s group had also reported that antibody 7D10 recognizes the N-terminal domain of MERS-CoV S glycoprotein, representing a new neutralizing epitope outside the RBD. Their study further implied that 7D10 has a different neutralizing mechanism; it mainly interferes with the pre- to postfusion conformational changes of S glycoprotein required for membrane fusion. Collectively, their work provides critical information for understanding the host-antibody response and for guiding the combined application of different antibodies for defense against MERS-CoV infection.

References1. D. Wang et al., Nat. Immunol. 11, 905–911 (2010).2. H. Wei et al., FEBS Lett. 588, 3838–3843 (2014).3. X. Liu et al., Proc. Natl. Acad. Sci. U. S. A. 110,

14918–14923 (2013).4. J. Ge et al., Structure 27, 1296–1307 (2019).5. N. Wang et al., Cell. Res. 23, 986–993 (2013).6. M. Gui et al., Cell. Res. 27, 119–129 (2017).7. W. Song, M. Gui, X. Wang, Y. Xiang, PLOS

Pathog. 14, e1007236 (2018).8. H. Zhou et al., Nat. Commun. 10, 3068 (2019).9. S. Zhang et al., Cell Rep. 24, 441–452 (2018).10. X. Yu et al., Sci. Rep. 5, 13133 (2015).11. P. Niu et al., J. Infect. Dis. 218, 1249–1260 (2018).12. L. Jiang et al., Sci. Transl. Med. 6, 234ra59

(2014).

FIGURE 2. Structural snapshots of SARS-CoV spike (S) glycoprotein trimer in different states during receptor binding and membrane fusion. Binding of ACE2 to trimeric S glycoprotein in the active state triggers conformational changes required for membrane fusion, including the dissociation of S1-ACE2 from the S trimer and rearrangement of fusion peptide (FP), heptad repeat 1 (HR1), and heptad repeat 2 (HR2) in the S2 subunit.

FIGURE 3. Epitopes and mechanisms of representative MERS-CoV neutralizing mAbs MERS-4, MERS-27, MERS-GD27, and 7D10.

The Research Group of Ye XiangYe Xiang

Ye Xiang received his bachelor ’s degree from the College of Chemistry and Molecular Engineering at Peking University in 1998 and his doctorate from the Institute of Biophysics at the Chinese Academy of Sciences (CAS) in 2003. He pursued his postdoctoral training in the laboratory of Michael G. Rossmann at Purdue University, Indiana, between 2005 and 2012, where he studied the molecular mechanisms involved in the infection and assembly of bacteriophage ϕ29. In 2012, Xiang obtained funding support from the government of China. In 2013, he returned to Tsinghua University in Beijing as an assistant professor to initiate his independent

research. He was promoted to the position of a tenured associate professor at the end of 2017.

Xiang’s research group currently has 10 graduate students. The group's long-term goal is to understand the molecular mechanisms underlying virus assembly and infection. The group mainly uses biochemical and biophysical approaches for their studies. To date, the Xiang group has published more than 15 scientific articles in prestigious journals, including Science, Nature, Molecular Cell, Nature Communications, and PNAS. The major contributions made by Xiang’s research team are summarized as follows:

1. Uncovering the membrane penetration mechanisms of tailed bacteriophages

Viruses must breach the host-cell membrane for successful infection. The mechanisms involved in the host membrane penetration by most tailed prokaryotic viruses (also known as tailed bacteriophages) are not completely clear.

The tailed bacteriophage ϕ29 is a well-studied model system for understanding virus infection and assembly. The distal end of its tail is an enlarged cylindrical structure called the tail knob, which is

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Medicine,�Tsinghua�University,�Beijing,�[email protected]�


largely a protein assembly of gp9. Structural studies of the ϕ29 tail knob protein gp9 by Xiang’s group have shown that the pseudo sixfold cylindrical tube of the tail knob is constituted of six gp9 molecules arranged as three dimers. The distal end of the tube is blocked by six long hydrophobic gp9 loops, which are rich in glycine and share weak similarities to those of viral fusion peptides observed in eukaryotic viruses. Further studies have shown that the hydrophobic loops exit during virus infection and are inserted into the host-cell membrane lipid bilayer to form a pore structure for the translocation of the viral genome. The combined data suggested a membrane-active, peptide-associated, pore-forming mechanism of prokaryotic viruses for overcoming the membrane barrel (1, 2).

2. Molecular mechanisms involved in the assembly of tailed bacteriophages

The assembly of tailed bacteriophages is complex and includes several key steps. Mediated by a small scaffolding protein, the empty head of a phage is first assembled. The genome of the virus is packed into the empty protein shell of the head by an ATP-consuming packaging motor formed around the portal complex of the head. The packaging motor is discarded once the head is full, through a yet unknown mechanism. Finally, the tail components sequentially assemble on the head, forming the mature phage. The mechanisms involved in the assembly of tailed bacteriophages are

largely unknown. With the help of the model system ϕ29, Xiang’s group studied the atomic structures of several-megadalton key ϕ29 particles (3) (Figure 1). Structural analysis and comparisons revealed that at the last stage of genome packaging, accumulation of pressure in the head pushes the portal complex out of the head to discard the packaging motor and terminate genome packaging. The 12 tail appendages of ϕ29, in response to host-cell receptor recognition, form an interlock assembly around the tail neck, suggesting that the appendages may function synergistically in sensing host-cell receptors. Release of the genome during infection is controlled by the tail tube assembly, which has a unique distribution of charged residues inside. Together, these results revealed the molecular details of the assembly of a tailed bacteriophage (3).

3. Molecular mechanisms involved in the host-cell entry of enveloped eukaryotic viruses

The spike (S) glycoprotein of severe acute respiratory system (SARS) coronavirus (SARS-CoV) plays an essential role in virus entry; it attaches to the host cell surface by binding to specific host-cell receptors and subsequently mediates host-cell membrane and viral membrane fusion through striking conformational changes. In collaboration with Xinquan Wang’s group at the School of Life Sciences at Tsinghua University, Xiang’s group determined the structure of SARS-CoV S glycoprotein spike trimer in four different conformations (4). Structure analysis and comparisons showed that three conformations of the spike trimer are asymmetric, with one receptor-binding domain protruding up. The “up” conformation allows the binding of the virus receptor and is required for the binding of several well-characterized SARS-CoV neutralizing antibodies that recognize epitopes on the receptor binding domain. Further studies showed that upon binding to the receptor, the “up” receptor-binding domain opens up and triggers the pre- to postfusion transition of the spike (5). These results together established complete snapshots of the SARS-CoV spike during infection (Figure 2).

References1. J. Xu et al., Nature 534, 544–547 (2016).2. J. Xu, Y. Xiang, J. Virol. 91, e00162–17 (2017).3. J. Xu et al., Nat. Commun. 10, 2366 (2019). 4. M. Gui et al., Cell Res. 27, 119–129 (2017). 5. W. Song et al., PLOS Pathog. 14, e1007236 (2018).

FIGURE 1. Atomic structures of the three key particles assembled in the life cycle of bacteriophage ϕ29.

FIGURE 2. A schematic diagram showing the molecular mechanisms involved in SARS coronavirus entry.

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The Research Group of Pilong LiJing Wang

Pilong Li received his bachelor ’s degree from Peking University in 2000 and his doctorate from the University of Texas Southwestern (UTSW) Medical Center in 2009. He pursued his postdoctoral training at the University of Pennsylvania between 2010 and 2015. At UTSW, Li was involved in in vitro reconstitution of liquid–liquid phase separation (LLPS) from simple biochemical systems (1). LLPS has been proven to be a general molecular mechanism underlying numerous biomolecular condensates in cells. In 2016, Li joined Tsinghua University as a tenure-track assistant professor.

The Li laboratory consists of three staff members, three postdoctoral fellows, and 12 graduate students. The group combines biophysics, biochemistry, and cell biology to elucidate the physiological and pathological roles of LLPS in various fundamental pathways. To date, Li’s group has published five research papers in Nature, Cell Research, and Molecular Cell. The major contributions of the Li group are summarized below.

1. Elucidation of the mechanism of cargo segregation in p62-mediated selective autophagy

Selective autophagy is an important pathway for maintaining cellular protein homeostasis by degrading polyubiquitinated proteins. Autophagic cargo proteins were previously believed to be concentrated into aggregates by the selective autophagy receptor p62, and the aggregates were considered to be subsequently degraded by autophagosomes. However, Li’s group and their collaborators demonstrated that polyubiquitin chains induce LLPS of p62 to form the p62 body, which drives autophagic cargo segregation (Figure 1) (2).

2. Role of LLPS in heterochromatin formation in animals and plants

Eukaryotic chromosomes contain distinct compartments, which are characterized by specific, posttranslational modifications of core histones. However, the molecular mechanisms by which these histone modifications function in chromosome compartmentalization remain poorly understood. Constitutive heterochromatin is a silent chromosome compartment characterized by H3K9me2/3. In animals, heterochromatin protein 1 (HP1) is an H3K9me2/3 reader. HP1 interacts with SUV39H1 (an H3K9me2/3 writer) and HP1 scaffolding protein to form complexes with multiple chromodomains, which are H3K9me2/3 reading modules. H3K9me3-marked nucleosomal arrays and HP1-containing

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�[email protected]�

FIGURE 1. A model depicting the formation of the p62 body via LLPS in selective autophagy.

FIGURE 2. H3K9me2/3-mediated phase separation is critical for heterochromatin organization in animals and plants .

FIGURE 3. FLL2 promotes phase separation of polyadenylation complexes in plants .


complexes undergo LLPS to form liquid-phase biomacromolecular condensates. The latter are reminiscent of heterochromatin in the nuclei (3). In plants, the functional counterparts of animal HP1 have remained elusive. Li’s group, along with their collaborators, recently identified the first plant H3K9me2/3 reader, ADCP1, which contains at least three H3K9me2/3 reader domains. ADCP1 undergoes LLPS with H3K9me3 nucleosomal arrays, similar to the HP1-containing complexes of animals (4). This property of ADCP1 is likely crucial for its role in heterochromatin organization in plants. Collectively, the group's studies have indicated H3K9me2/3-mediated LLPS to be an important mechanism underlying heterochromatin organization in animals and plants (Figure 2).

3. Identif ication of a coiled-coil protein capable of promoting phase separation of polyadenylation complexes in plants

Extensive in vitro studies have shown that multivalent interactions drive LLPS (1). However, the regulation of LLPS in vivo remains largely unknown. Li’s group and their collaborators identified FLL2, an in vivo regulator of LLPS of the Arabidopsis RNA-binding protein FCA. Furthermore, their studies have suggested that FLL2 is required for forming FCA nuclear bodies and for FCA to properly function in the 3' cleavage and polyadenylation of target RNAs. In addition, they have also uncovered the key components of phase-separated nuclear bodies—i.e., FLL2 and FCA—as well as 3' processing factors. Collectively, the findings revealed that the coiled-coil protein, FLL2, promotes the LLPS of FCA to form FCA nuclear bodies, which compartmentalize 3' processing factors for the cleavage and polyadenylation of target RNAs (Figure 3) (5).

4. Demonstrating the role of multivalent m6A motifs in promoting YTHDF phase separation

N6-methyladenosine (m6A) is an abundant histone modification on eukaryotic RNAs and plays an important role in multiple biological functions. To shed light on the molecular mechanism underlying the function of m6A in various pathways, Li’s group studied the interaction between m6A and its readers—the YT521-B homology domain family (YTHDF) proteins. They found three human YTHDF proteins (YTHDF1, YTHDF2, and YTHDF3) with low-complexity regions that possess intrinsic LLPS capacity. Importantly, they showed that multivalent m6A-containing RNAs promote the LLPS of YTHDF proteins, presumably by binding multiple protein molecules, both in vitro and in cells (Figure 4). Their work also suggested a plausible mechanism through which m6A and YTHDF2 may be involved in the stress response of cells (6).

The long-term goal of Li’s group is to reveal more phase separation–based cellular processes under both physiological and pathological conditions and to develop strategies to combat diseases caused by such aberrant phase separations.

References1. P. Li et al., Nature 483, 336–340 (2012).2. D. Sun et al., Cell Res. 28, 405–415 (2018).3. L. Wang et al., Mol. Cell 76, 1–14 (2019).4. S. Zhao et al., Cell Res. 29, 54–66 (2019).5. X. Fang et al., Nature 569, 265–269 (2019).6. Y. Gao et al., Cell Res. 29, 767–769 (2019).

FIGURE 4. Multivalent m6A promotes phase separation of YTHDF proteins.

The Research Group of Hang Huber t YinYing Zhang, Hang Yin*

Hang Hubert Yin obtained his bachelor ’s degree from Peking University and his doctorate from Yale University in 2004, under the supervision of Andrew Hamilton. He pursued his postdoctoral research at the Perelman School of Medicine, University of Pennsylvania, with William DeGrado. Prior to joining Tsinghua, he was a tenured faculty member at the University of Colorado, Boulder. Yin is currently a professor and deputy dean of the School of Pharmaceutical Sciences, Tsinghua University. Since he started his independent career in 2007, his group has published more than 100 papers in leading journals, including several in Science, Nature,

and Cell, with over 7,000 total citations. Moreover, he is a recipient of many accolades, including the American Chemical Society David W. Robertson Award for Excellence in Medicinal Chemistry, the National Science Fund for Distinguished Young Scholars Beijing Outstanding Young Scientist Prize, the Chinese-American Chemistry & Chemical Biology Professors AssociationDistinguished Junior Faculty Award, and the American Association for Cancer Research Gertrude B. Elion Cancer Research Award.

Yin’s research interests lie at the interface of chemistry, biology, and pharmaceutical science, with a particular focus on structure-based drug design, cell-signaling biochemistry, biotechnology development, and membrane biophysics. The major contributions made by Yin’s research team are summarized as follows:

1. Small-molecule immunomodulators targeting Toll-like receptors

Yin’s group has combined expertise in various fields to develop novel small-molecule regulators of Toll-like receptors (TLRs). His team has developed selective small-molecule regulators for TLR1/2

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Pharmaceutical�Sciences,�Tsinghua�University,�Beijing,�China*Corresponding�author:�[email protected]

29CHAPTER 3

complex, TLR3, TLR4, TLR5, and TLR8, as well as simultaneous activators for TLR3/8/9, all of which have significant potential in drug discovery (1–4) (Figure 1).

2. New understanding of innate immunity and pro-inflammatory signaling

Recently, Yin’s team has found that extracellular vesicles play important roles in the propagation of TLR9-mediated immune signals (5) (Figure 2). Previously, the team had discovered morphine-induced TLR4 activation to be a mechanism of opioid-based pain management and reported unexpected results implicating HMGB-1 as a danger-signal molecule that binds to TLR5 for proinflammatory signaling (6–7). Additionally, their report on caspases as a new pharmacological target for nonsteroidal anti-inflammatory drugs has implications for patient safety and next-generation drug design (8).

3. Development of chemical probes that target protein–protein interactions in transmembrane domains

Protein–protein interactions in the membrane are little understood due to the lack of exogenous agents with high specificity and selectivity. The team has developed a novel, generally applicable method, using the CHAMP (computed helical anti-membrane protein) program, for de novo designing of peptide antagonists against protein–protein interactions in the single-pass protein transmembrane region, which may be applied in the recognition of transmembrane helices of α-subunits of platelet integrins, αIIbβ3 and αvβ3 (9).

4. Curvature-sensing biotechnology by targeting protein–lipid interactions

Exosome-enabled biofluid diagnostics are potentially applicable to tumor metastasis and other diseases. Yin’s laboratory has successfully developed a number of promising peptides and peptidomimetic agents that can sense and/or induce membrane curvature, providing a potential measure of the concentration of exosomes in solution and blood plasma (10).

Taken together, the major focus of Yin’s laboratory is to develop technologies with potential biomedical applications. In the past 12 years, his group's efforts have led to four issued patents and nine additional patent applications. Current and future areas of research include systematic development of chemical probes targeting

endosomal TLRs, regulation of non-TLR innate immunity, further understanding of innate immune pathways and pharmacology of nonsteroidal anti-inflammatory drugs, and development of novel biotechnology techniques for extracellular vesicles (EVs).

References1. K. Cheng et al., J. Am. Chem. Soc. 133, 3764–3767 (2011).2. S. Zhang et al., Nat. Chem. Biol. 14, 58–64 (2018).3. K. Cheng et al., Sci. Adv. 1, (2015).4. K. Cheng et al., Angew. Chem. Int. Edit. 51, 12246–12249 (2012).5. Y. Zhang et al., Sci. Adv. 5, eaav1564 (2019).6. N. Das et al., Cell Rep. 17, 1128–1140 (2016). 7. X. Wang et al., Proc. Natl. Acad. Sci. U. S. A. 109, 6325–6330 (2012).8. C. E. Smith et al., Cell Chem. Biol. 24, 281–292 (2017).9. H. Yin et al., Science 315, 1817–1822 (2007).10. H. Yin et al., Annu. Rev. Biomed. Eng. 18, 51–76 (2016).

FIGURE 1. Small-molecule regulators of Toll-like receptors developed by Yin’s team.

FIGURE 2. The transportation of oligodeoxynucleotide (ODN) and Cdc42 from TLR9-activated macrophages to naïve cells via extracellular vesicles (EVs) exerted synergetic effects in the propagation of intracellular immune response (5). CC BY-NC (https://creativecommons.org/licenses/by-nc/4.0/).


The Research Group of Xu TanXu Tan

Xu Tan is an associate professor at the School of Pharmaceutical Sciences at Tsinghua University. He obtained his bachelor ’s degree in biology with honors from the University of Science and Technology of China in 2003 and his doctoral degree in pharmacology from the University of Washington in 2007. In his doctoral studies under the direction of Ning Zheng, Tan elucidated the structural mechanisms underlying the plant-sensing activity of two key plant hormones, auxin and jasmonate. In addition, Tan conducted his postdoctoral research on antiviral drug screening with Stephen Elledge at Harvard Medical School from 2008 to 2014. In 2014, Tan established his laboratory at Tsinghua University to conduct research on virus-host interactions, antiviral drug discovery, and the ubiquitin-proteasome pathway, using genetic, biochemical, and structural approaches.

Xu Tan is the recipient of the GE & Science Prize for Young Life Scientists (2008, North America), the Harold M. Weintraub Graduate Student Award (2008), the Damon Runyon Postdoctoral Fellowship Award (2012), and the Young Scientist Award of the National Natural Science Foundation of China (2017). The major scientific contributions of the Tan laboratory are summarized below.

1. Identif ied a new HIV restriction factor, PSGL-1, and elucidated its antiviral mechanism.

PSGL-1 potently inhibits the infectivity of HIV virions by being incorporated into the virions. Tan and colleagues discovered the antagonistic strategy by which HIV uses its accessory protein Vpu to hijack SCF-βTrCP2 ubiquitin ligase from the host to promote PSGL-1 ubiquitination and degradation. The antagonism between HIV Vpu and PSGL-1 provides a potential target for developing new HIV drugs (1) (Figure 1).

2. Developed a new delivery method for in vivo gene therapy.Tan and colleagues developed an in vivo gene-therapy delivery

method based on the CRISPR-Cas9 gene-editing system and demonstrated its efficacy in suppressing hepatitis B virus replication and inhibiting the liver protein PCSK9, a therapeutic target for hypercholesterolemia (2). To achieve in vivo delivery of the CRISPR-Cas9 system, this method used novel lipid-nanoparticle material that is free of the traditional virus vectors, thereby providing a potentially safer alternative approach for gene therapy.

3. Identif ied a novel cause of a rare genetic skin disorder called Epidermolysis bullosa and the mechanism for keratin protein degradation.

The Tan laboratory discovered a new disease-causing mechanism of ubiquitin ligase KLHL24 due to the loss of its autoregulatory

mechanism. This work also identified the first ubiquitin ligase to directly regulate keratin protein turnover (3).

4. Developed a drug screening method for eff icient and systematic identif ication of synergistic drug combinations and discovered novel anti-HIV compounds (4).

5. Uncovered the structural mechanism for receptors of the plant hormones auxin and jasmonate, identif ied inositol polyphosphates as cofactors of the two receptors, and established a novel model for hormone action (5, 6).

References1. Y. Liu et al., Nat. Microbiol. 4, 813–825 (2019).2. C. Jiang et al., Cell Res. 27, 440–443 (2017).3. Z. Lin et al., Nat. Genet. 48, 1508–1516 (2016).4. X. Tan et al., Nat. Biotechnol. 30, 1125–1130 (2012).5. L. B. Sheard et al., Nature 468, 400–405 (2010).6. X. Tan et al., Nature 446, 640–645 (2007).

FIGURE 1. New restriction factor for HIV infection: PSGL-1 (red) is incorporated into nascent HIV virions and inhibits their infectivity (image design by Dali Wu) (1).


31CHAPTER 4

The Research Group of Haitao LiHuida Ma and Xingrun Zhang*

Haitao Li received his bachelor ’s degree in microbiology from Shandong University in 1997 and his doctorate degree in molecular biophysics at the Institute of Biophysics, Chinese Academy of Sciences, in 2003. He then joined the laboratory of Dinshaw J. Patel at Memorial Sloan Kettering Cancer Center to carry out his postdoctoral research in epigenetic regulation. Li joined the School of Medicine at Tsinghua University as a tenure-track associate professor in 2010 and was promoted to full professor with tenure in 2016. Li currently serves as associate director of the Beijing Advanced Innovation Center for Structural Biology and associate dean of the Department of Basic Medical Sciences, School of Medicine, Tsinghua University. He is the recipient of numerous awards, including the Promega Innovation Award for Cell Biology, the Young Scholar of China Award in Cancer Research, the Wuxi PharmaTech Life Science and Chemistry Awards, and the HFSP Young Investigators Grant Award, as well as a grantee of the National Science Fund for Distinguished Young Scholars of China.

Li’s research is focused on gaining a molecular understanding of epigenetic regulation and modification biology. Epigenetics concerns the process by which genetic information is organized and decoded at the chromosomal level and is now recognized to play a key role in many biological processes, ranging from gene regulation to cell fate decisions. Moreover, accumulating evidence points to an association between epigenetic dysregulation and diverse human diseases, notably cancer. Li’s research group consists of one assistant professor, three research assistants, three postdoctoral fellows, and 15 doctoral students. The group combines structural, biochemical/chemical, and cellular approaches to study the key recognition and catalysis events involved in epigenetic regulation. Other endeavors of the lab include structure-guided drug discovery and biointeraction-profiling tool development.

Li’s major contribution to science has been the elucidation of the molecular basis underlying modification-dependent histone/DNA recognition and catalysis by so-called “readers/writers/erasers” and their impact on health and disease. Examples include

the PHD finger, MBT, ADD, YEATS, Bromo, PWWP, Spin/Ssty, DPF, BAH, Agenet, NRMT1, SETD2, SIRT3, and ALKBH1. Since 2010, the Li group has published over 60 research or review articles in prestigious journals, including Nature, Cell, Cell Research, Molecular Cell, Nature Chemical Biology, and Genes and Development. The major research achievements of the Li group are summarized below.

1. Characterization of YEATS domains as a novel family of histone acetylation readers

Recognition of histone posttranslational modifications constitutes a key mechanism for gene regulation in a chromatin context (1, 2). In collaboration with Xiaobing Shi at the University of Texas MD Anderson Cancer Center (currently a professor at the Van Andel Institute), the Li group characterized the immunoglobin-fold YEATS domain as a novel class of acetyl-lysine binding modules (Figure 1), and established a direct link between the histone H3K9 acetylation readout and DOT1L-mediated H3K79 methylation in transcription control (3). There are four human YEATS proteins—namely, AF9, ENL, GAS41, and YEATS2—which are all implicated in important cellular processes, and their dysfunctions are closely linked to human diseases such as leukemia and glioblastoma. Through structural and binding analyses, the Li group defined ENL and AF9 as histone H3K9, H3K18, and H3K27 acetylation readers (4), YEATS2 as an H3K27ac-specific reader (5), and Gas41 as a reader for both H3K27ac and H3K14ac (6, 7). These efforts established the molecular basis underlying the histone acetylation–specific readout by human YEATS proteins. Moreover, inspired by the fact that YEATS shares a similar immunoglobin fold with that of an antibody, Li proposed the “antibody-like loop evolution” hypothesis for the molecular design of readers by which a reader pocket is evolved to recognize a mark. This mechanism is similar to the rearrangement of the complementarity-determining region loops of an antibody for recognition of an antigen with minimal perturbation of overall folding (8). In collaboration with the laboratories of Xiaobing Shi, Scott Armstrong, David Allis, and Xiang David Li, the Li group further investigated the functional roles of YEATS proteins in tumorigenesis and stem cell biology (3–7), and successfully developed the first-in-class YEATS inhibitor of ENL/AF9 (9).

2. Non-acetyl histone acylation readout and catalysis : linking metabolism to epigenetics

Histone acylation constitutes a crucial epigenetic mechanism that regulates diverse biological processes by bridging metabolism and epigenetics (10). The development of advanced proteomics

FIGURE 1. Discovery of the YEATS domain as a novel family of histone acylation readers. Kac, lysine acetylation; Kcr, lysine crotonylation; acyl, acylations. The immunoglobin fold of AF9 YEATS is depicted as a red ribbon. Note the formation of an acyl-binding pocket at the loop regions. AF9, ENL, GAS41, and YEATS2 are four human YEATS proteins.

Beijing�Advanced�Innovation�Center�for�Structural�Biology,�Department��of�Basic�Medical�Sciences,�School�of�Medicine,�Tsinghua�University,�Beijing,�China*Corresponding�author:�[email protected]


technologies led to the identification of diverse non-acetyl histone acylations, including propionylation, butyrylation, crotonylation, succinylation, malonylation, glutarylation, β-hydroxybutyrylation, and benzoylation, posing new challenges to identify their cognate regulators (11). In 2016, the Li group first demonstrated that the YEATS domains are better readers for histone crotonylation than histone acetylation, and promote transcription in response to metabolic alternations (12, 13). Subsequently, they characterized the DPF domains as a second class of preferential histone crotonylation readers (14). The discovery of two classes of favorable crotonylation readers highlighted a new regulatory mechanism based on non-acetyl histone acylation readout by the YEATS and DPF family proteins (Figure 2). In 2019, the Li group characterized human SIRT3 as a histone deacylase that acts on non-Gly-flanking β-hydroxybutyrylated lysines (Kbhb) (15). Such class selectivity is unique to NAD+-dependent sirtuins but is not exhibited for Zn2+-dependent HDAC family members owing to the β-backbone substrate recognition mode of sirtuins. This work sheds light on the function of sirtuins in Kbhb and acylation biology through hierarchical deacylation (Figure 2). In recognition of Li’s scientific contributions, he was invited to write three reviews on histone acylation biology (8, 11, 16).

3. Multivalent engagement , phase separation, and modification crosstalk

Histone modifications often exist as a recognizable pattern, and the reader modules are frequently linked in pairs, suggesting a mechanism of multivalent engagement. In 2007, Li proposed the hypothesis of combinatorial readout and multivalent engagement along with David Allis and Dinshaw J. Patel (1, 17). To experimentally

verify this hypothesis, Li characterized a series of multivalent engagement events (Figure 3A), including a nucleosomal histone “H3K4me3-H4K16ac” readout by BPTF (18), histone H3 “K4me0-K9me3” readout by ATRX (19), H3 “K4me3-R8me2a” methylation pattern readout by Spindlin1 (20), histone variant H3.3 “S31-K36me3” readout by ZMYND11 (21), H3 “K4me0/1-K14ac” readout by ZMYND8 (22), H3 “R2me0-K4me0-K14cr” readout by MOZ (14), and H3K9me2/3-modified nucleosome array engagement by plant ADCP1, which is a functional homolog of animal HP1 (23). Remarkably, one of the less-appreciated consequences of multivalent engagement is liquid–liquid phase separation (24). In collaboration with the research groups of Pilong Li and Qianwen Sun at Tsinghua University School of Life Sciences, the Li group demonstrated that the multivalent engagement of chromatin H3K9 methylation by plant ADCP1 and animal HP1 could trigger the formation of a heterochromatic condensate through a phase-separation mechanism (Figure 3B) (23, 25).

In addition to an avidity effect associated with combinatorial readout, binary switch and bivalent tolerance are two other consequences of modification interplay (Figure 3C). The Li group demonstrated the binary switch effects of H3S10ph against the recognition pair “H3K9me2/3-ADCP1/HP1” (23, 25), H3T3ph against “H3K4me0-DNMT3A” (26), H3S28ph against “H3K27me3-BAHD1” (27), and H3.3S31ph against “H3.3K36me3-ZMYND11” (21). Moreover, they revealed H3S10ph tolerance by “H3K9me3-ATRX” (28), and “H3T3ph,” “H3K9me3,” and “H3K9me3-S10ph” tolerance by “H3K4me0-Sp100c” (29) binding pairs.

Among these interactions, ZMYND11 and ZMYND8 are newly identified epigenetic tumor suppressors (21, 22); Spindlin1 and MOZ are known as epigenetic tumor promoters (14, 20); and ATRX,

FIGURE 2. Linking metabolism and epigenetics through histone acylation and its regulators . Metabolic intermediates can be imported into the nucleus to modify histones by a series of acyl-CoA-dependent acyl-transferases such as p300 and GCN5. Histone crotonylation (Kcr) is a better mark than histone acetylation (Kac) for YEATS and DPF but not for Bromo, which differentiates the three classes of Kac readers. SIRT3 and HDAC3 are representative members of the NAD+-dependent sirtuin and Zn2+-dependent HDAC family of acylation erasers, respectively. Unlike HDAC3, SIRT3 cannot remove acylation marks with flanking glycine residues such as histone H4K5, H4K8, and H4K12.

33CHAPTER 4

DNMT3A, HP1, BAHD1, and Sp100c are often associated with a silencing function and regulate cellular processes such as immune responses and heterochromatin formation (25, 27, 29, 30). Hence, continued research by the Li group on multivalent engagement can provide critical mechanistic insights into posttranslational, modification-mediated chromatin regulation in health and disease.

4. Histone N-terminal methylation by NRMT1 and oncohistone recognition by SETD2

Methylation often serves as a biochemical strategy to add new traits for recognition and regulation through modification of residues. Besides side-chain methylations, the α-amine group can also be methylated by a family of N-terminal methyl transferases. The Li group reported high-resolution cocrystal structures of human NRMT1 bound to human histone CENP-A and fruit fly histone H2B (31). This work provided the first insights into the molecular basis underlying histone substrate recognition and trimethyl state-specific catalysis by NRMT1.

High-frequency point mutations of genes encoding histones have been recently identified as novel drivers in several tumors. Specifically, the H3K36M/I and H3G34R/V mutations were shown to be oncogenic in bone and brain tumors by inhibiting H3K36 methyltransferases, including SETD2. The Li group performed cocrystal structural and enzymatic studies of SETD2, and revealed a trans-inhibitory role of H3K36M/I and a cis-inhibitory role of H3G34R/V in suppressing SETD2 activity (32). This work revealed

the molecular basis underlying the oncohistone recognition by SETD2 and its inhibition.

5. Deep-profiling tool development for epigenetics and modification biology

Epigenetic regulation relies heavily on modification-dependent recognition and catalysis. Compounding this complexity, extensive nonhistone and RNA modifications have been shown to exert important functions on par with those of histone/DNA modifications (33). Hence, there is an urgent need to establish a platform for high-throughput and quantitative biointeraction profiling, especially for modification-dependent interactions (34). To this end, in collaboration with Jinsong Zhu at the Chinese National Center for Nanoscience and Technology, the Li group developed a three-dimensional (carbene-chip) surface plasmon resonance imaging (3D-SPRi) platform for lab-on-chip profiling of epi-interactions (Figure 4A) (35). The 3D-SPRi technology features the advantages of real-time, quantitative, label-free, and high-throughput screening of immobilized peptides, DNA, RNA, proteins, and drug molecules (Figure 4B). The Li group has used this platform to successfully profile hundreds of plant readers (Figure 4C) (36) and discovered the long-sought-after plant HP1 protein–ADCP1 (23). Conceivably, such a deep-profiling platform will pave the way for new discoveries centered on biological interactions in epigenetics and beyond.

As an extension of the central dogma of molecular biology, dynamic modifications of macromolecules play a key role in shaping

FIGURE 3. Multivalent engagement , phase separation, and modification crosstalk . (A) Combinatorial readout of histone modification patterns by paired reader modules. (B) ADCP1 mediation of liquid–liquid phase separation in an H3K9 methylation-dependent manner. (C) Adjacent histone modification crosstalk can be involved in avidity, bivalent tolerance, and binary switch effects. All examples are based on research results from the Haitao Li group.


cellular traits through modulating diverse cellular processes including, but not limited to, epigenetic regulation. Thus, one of the long-term goals of the Li group is to unravel the working mechanisms from a broader sense of modification biology and to develop new therapeutic strategies for treating modification-related diseases.

References 1. S. D. Taverna, H. Li, A. J. Ruthenburg, C. D. Allis, D. J. Patel, Nat. Struct. Mol. Biol. 14,

1025–1040 (2007).2. R. G. Roeder, Nat. Struct. Mol. Biol. 26, 783–791 (2019).3. Y. Li et al., Cell 159, 558–571 (2014).4. L. Wan et al., Nature 543, 265–269 (2017).5. W. Mi et al., Nat. Commun. 8, 1088 (2017).6. C. C. Hsu et al., Cell Discov. 4, 28 (2018).7. C. C. Hsu et al., Genes Dev. 32, 58–69 (2018).8. D. Zhao, Y. Li, X. Xiong, Z. Chen, H. Li, J. Mol. Biol. 429, 1994–2002 (2017).9. X. Li et al., Nat. Chem. Biol. 14, 1140–1149 (2018).10. B. R. Sabari, D. Zhang, C. D. Allis, Y. Zhao, Nat. Rev. Mol. Cell Biol. 18, 90–101 (2017).11. S. Zhao, X. Zhang, H. Li, Curr. Opin. Struct. Biol. 53, 169–177 (2018).12. D. Zhao et al., Cell Res. 26, 629–632 (2016).13. Y. Li et al., Mol. Cell 62, 181–193 (2016).14. X. Xiong et al., Nat. Chem. Biol. 12, 1111–1118 (2016).15. X. R. Zhang et al., Cell Discov. 5, 35 (2019).

16. Y. Li, D. Zhao, Z. Chen, H. Li, Transcription 8, 9–14 (2017).17. A. J. Ruthenburg, H. Li, D. J. Patel, C. D. Allis, Nat. Rev. Mol. Cell Biol. 8, 983–994

(2007).18. A. J. Ruthenburg et al., Cell 145, 692–706 (2011).19. S. Iwase et al., Nat. Struct. Mol. Biol. 18, 769–776 (2011).20. X. Su et al., Genes Dev. 28, 622–636 (2014).21. H. Wen et al., Nature 508, 263–268 (2014).22. N. Li et al., Mol. Cell 63, 470–484 (2016).23. S. Zhao et al., Cell Res. 29, 54–66 (2019).24. P. Li et al., Nature 483, 336–340 (2012).25. L. Wang et al., Mol. Cell 76, 646–659.e6 (2019). 26. K. M. Noh et al., Mol. Cell 59, 89–103 (2015).27. D. Zhao et al., Protein Cell 7, 222–226 (2016).28. K. M. Noh et al., Proc. Natl. Acad. Sci. U. S. A. 112, 6820–6827 (2015).29. X. Zhang, D. Zhao, X. Xiong, Z. He, H. Li, J. Biol. Chem. 291, 12786–12798 (2016).30. K. M. Noh, C. D. Allis, H. Li, ACS Chem. Biol. 13, 1103 (2018).31. R. Wu, Y. Yue, X. Zheng, H. Li, Genes Dev. 29, 2337–2342 (2015).32. S. Yang et al., Genes Dev. 30, 1611–1616 (2016).33. E. M. Michalak, M. L. Burr, A. J. Bannister, M. A. Dawson, Nat. Rev. Mol. Cell Biol. 20,

573–589 (2019).34. S. Zhao, Y. Yue, Y. Li, H. Li, Curr. Opin. Chem. Biol. 51, 57–65 (2019).35. S. Zhao et al., Proc. Natl. Acad. Sci. U. S. A. 114, E7245–E7254 (2017).36. S. Zhao, B. Zhang, M. Yang, J. Zhu, H. Li, Cell Rep. 22, 1090–1102 (2018).

FIGURE 4. Development and application of the three-dimensional (3D)-SPRi platform for lab-on-chip biointeraction profiling. (A) Working scheme of 3D-carbene chip-based surface plasmon resonance imaging (3D-SPRi) technology. (B) Application of the 3D-SPRi platform for peptide and DNA/RNA oligo array screening to detect modification- and sequence-dependent recognition events. (C) Representative example of 3D-SPRi-based protein array screening and validation for biointeraction discovery in plant epigenetics.

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FIGURE 1. Summary of the major scientif ic contributions of Zhucheng Chen’s group. Bracketed numbers in image refer to references listed at the end of this paper.

The Laboratory of Zhucheng ChenZhucheng Chen

Zhucheng Chen received his doctorate from the Weill Cornell Graduate School of Medical Science in 2009. Thereafter, he pursued his postdoctoral training at the University of Texas Southwestern Medical Center in Dallas. In 2011, Chen joined the School of Life Science at Tsinghua University and was promoted to tenured associate professor in 2017. Chen was selected for the Thousand Young Talents program in 2012 and the National Science Fund for Distinguished Young Scholars in 2018.

Chen’s group comprises one postdoctoral fellow, 12 graduate students, and two research assistants. The group primarily focuses on chromatin biology, particularly chromatin remodeling, using various tools, including biochemistry, crystallography, cryo-electron microscopy (cryo-EM), and single-molecule fluorescence resonance energy transfer. Their major contributions are summarized below and in Figure 1.

1. The fundamental mechanism underlying chromatin remodelingNucleosomes are the basic repeating unit of chromatin.

Chromatin remodelers, including four major subfamilies (SWI/SNF, ISWI, CHD, and INO80), couple ATP hydrolysis to alter nucleosome positions and composition, thereby regulating chromatin structure and gene transcription.

To understand the mechanisms underlying chromatin remodeling, Chen’s group first determined the crystal structure of Snf2, the catalytic subunit of SWI/SNF, in the absence of the nucleosome (Figure 2) (1). Their study provides structural insights into the functional elements of Snf2 (Figure 2B). Thereafter, they determined the cryo-EM structure of Snf2 bound to the nucleosome (Figure 2C) (2), providing the first high-resolution view of the remodeler-DNA interaction. Furthermore, Zhucheng’s group determined that the cryo-EM structures of Snf2 bound to the nucleosome in the presence of different nucleotides (3). These structures revealed the

opened-closed conformational switch in the enzyme and the associated DNA translocations in an ATPase cycle. In particular, their findings indicate that ATP hydrolysis is coupled with the induction of local DNA bulges and long-range DNA translocations (Figure 2D). This structure reveals a new remodeling intermediate, thus furthering the current understanding of chromatin remodeling. Together, Chen’s group proposes a “DNA wave” model to explain the fundamental mechanism of DNA translocation underlying chromatin remodeling (Figure 2E).

2. Nucleosomal epitopes as the underlying mechanism regulating ISWI

While different chromatin remodelers share conserved catalytic cores, they are regulated distinctly and display functional diversity. Apart from Snf2, ISWI is highly regulated by nucleosomal epitopes to ensure an appropriate chromatin landscape in cells (Figure 3A).

To investigate the underlying mechanism regulating ISWI, Chen’s group elucidated the crystal structures of ISWI alone (Figure 3B) and determined that the complex bound to an H4 peptide (Figure 3C) (4). These findings reveal the mechanism underlying ISWI autoinhibition and provide insights into ISWI activation by H4 tails and by extranucleosomal linker DNA. To investigate the ISWI–nucleosome interaction, Chen’s group determined the cryo-EM structures of the ISWI–nucleosome complex (Figure 3D) (5) and found that the ISWI-bound DNA adopts the same conformations as Snf2-bound DNA (Figure 3E). These findings illustrate the mechanism of action of ISWI and its regulation through environmental clues (Figure 3F).

In sum, Chen’s group has described a unified chromatin remodeling mechanism modulated by nucleosomal epitopes.

3. Other achievements Chen’s group reported the first crystal structure of nuclear actin

(N-actin) bound to Arp4 and the HSA-helix of the chromatin-remodeling enzyme Swr1 (6). As a key cytoskeletal component, the presence of actin inside the nucleus was once controversial. However, actin is now considered a constitutive subunit of many chromatin-remodeling complexes. The crystal structure of N-actin, determined by Chen’s group, reveals the salient features of N-actin. This study provides a basis for studies on N-actin in the field of chromatin remodeling.

Beijing�Advanced�Innovation�Center�for�Structural�Biology�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�[email protected]


FIGURE 2. The mechanism underlying chromatin remodeling. (A) Domain organization of Snf2. (B) Crystal structure of Snf2. (C) Cryo-electron microscopic structure of the Snf2-nucleosome complex. The primary nucleosome-binding site is enlarged for further analysis on the right. (D) Local DNA bulge and long-ranged DNA translocation induced by Snf2 in the ADP-bound state. Bound nucleosome, color-coded; unbound nucleosome, grey. Bound Snf2 is schematically illustrated as an oval shape. The bottom panel depicts a schematic representation of DNA translocation around SHL2. (E) A simplified model of chromatin remodeling. This figure is modified from (1–3).

FIGURE 3. Structure and regulation of ISWI. (A) Domain organization and regulation of ISWI. (B) Crystal structure of ISWI. (C) Superimposition of the structures of ISWI (cyan) in the H4 (yellow)–bound and auto-inhibited (gray) states. (D) The overall structure of nucleosome-bound ISWI in the presence of ADP. (E) Comparison of the structures of the nucleosome in the unbound state (gray) and bound to ISWI (in the ADP state, color-coded). (F) Models for ISWI function and regulation. This figure is modified from (4, 5).

Chen’s group revealed the crystal and cryo-EM structures of the catalytic subcomplex of the histone acetyltransferase NuA4 (7). NuA4 catalyzes the acetylation of histone H4. However, the mechanism underlying nucleosomal histone recognition by NuA4 has remained unknown. Zhucheng’s group reported a novel mechanism underlying space-sequence double recognition of the H4 tails. Unlike the common mechanism underlying sequence-based recognition, this position-based mechanism of NuA4 provides novel insights into the action of histone acetyltransferase on the nucleosome substrate.

The long-term goal of Chen’s group is to reveal the molecular mechanisms underlying nucleosome-based phenomena and

to develop strategies to treat diseases via a chromatin biology/epigenetics approach.

References1. X. Xia et al., Nat. Struct. Mol. Biol. 23, 722–729 (2016).2. X. Liu et al., Nature 544, 440–445 (2017).3. M. Li et al., Nature 567, 409–413 (2019).4. L. Yan et al., Nature 540, 466–469 (2016).5. L. Yan et al., Nat. Struct. Mol. Biol. 26, 258–266 (2019).6. T. Cao et al., Proc. Natl. Acad. Sci. U. S. A. 113, 8985–8990 (2016).7. P. Xu et al., Mol. Cell 63, 965–975 (2016).

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The Laboratory of Chunlai ChenChunlai Chen

Chunlai Chen received his bachelor ’s degree in chemistry from the College of Chemistry and Molecular Engineering, Peking University, in 2003, and his doctorate in chemical biology from Peking University in 2008. Thereafter, he pursued a postdoctoral stint with Yale E. Goldman and Barry S. Cooperman at the University of Pennsylvania from 2008 to 2015. In 2015, Chen joined the School of Life Sciences at Tsinghua University to establish his independent group.

Chen’s group currently comprises eight graduate students and one staff member; they develop single-molecule fluorescence tools, combining them with other biochemical and biophysical approaches, to investigate the structural dynamics of biomolecules and to elucidate their mechanisms of action. The major contributions of Chen’s group, after joining the Beijing Advanced Innovation Center for Structural Biology, are the following:

1. Development of single-molecule f luorescence toolsSingle-molecule photoactivation fluorescence resonance energy

transfer (sm-PAFRET)Single-molecule fluorescence resonance energy transfer (sm-

FRET) is a widely used method for the elucidation of molecular mechanisms by providing dynamic information buried under ensemble-average measurements. However, concentrations of fluorescent species are restricted to ~50 nM or below, which is significantly lower than the binding constants of many biological processes under physiological conditions (>1 μM). Therefore, Chen’s group developed a method called sm-PAFRET, using photoactivatable fluorophores as donors to develop a general approach to disrupt the concentration barrier. sm-PAFRET has been used to elucidate novel pathways and capture transient FRET states during translation with ~1 μM of labeled elongation factor G. This concentration of elongation factor G is similar to its physiological concentration in cells and greater than the concentration of labeled elongation factor G used in previous sm-FRET measurements. In summary, Chen’s group reported that sm-PAFRET is an easy-to-implement tool for single-molecule FRET measurements and for detection of dynamic processes and molecular mechanisms at physiological concentrations (1, 2).

Surface transient binding-based fluorescence correlation spectroscopy (STB-FCS)

Fluorescence correlation spectroscopy (FCS) obtains information about the kinetic processes of freely diffusing biomolecules on the nanosecond timescale. However, its detection window is limited to submilliseconds, restricted by the dwell time of freely diffusing molecules in the laser excitation volume. To overcome this restriction, Chen’s group developed a simple and easy-to-use method called STB-FCS. Through transient interactions between immobilized probes and freely diffusing molecules, STB-FCS increases the dwell time of the molecules of interest in the excitation volume, extending the upper limit of the detection window to several seconds. Furthermore, for both intramolecular and intermolecular

reactions, two kinetic processes with relaxation time constants three orders of magnitude apart can be captured simultaneously via STB-FCS (3).

2. Capturing of structural dynamics and elucidation of molecular mechanisms

Cas9A major challenge for the application of Cas9 in genome editing

is off-target binding and cleavage. Using intramolecular sm-FRET measurements, Chen’s group revealed that Cas9 spontaneously fluctuates among three major conformational states in its apo, sgRNA-bound, and dsDNA/sgRNA-bound forms, emphasizing the conformational flexibility of the catalytic HNH domain. Furthermore, they uncovered a long-range allosteric interaction between the RNA/DNA heteroduplex at the PAM-distal end and the HNH domain, which ensures correct positioning of the catalytic site and serves as the final proofreading step of Cas9 before cleaving DNA. Several Cas9 residues were shown to mediate this allosteric interaction and proofreading step. Mutagenesis at these sites to modulate interactions between Cas9 and the heteroduplex at the distal end could potentially improve Cas9 specificity (4).

Bacterial ribosomeDownstream stable mRNA secondary structures can stall

ribosomal elongation by impeding translocation, which refers to concerted movements of transfer RNAs (tRNAs) and messenger RNAs (mRNAs) in the ribosome. Furthermore, to induce –1 programmed ribosomal frameshifting (–1 PRF), a downstream stem-loop or a pseudoknot mRNA structure is needed. Interestingly, –1 PRF efficiencies are correlated with the probabilities of pseudoknots to form alternative structures but not with the mechanical properties of pseudoknots. Based on sm-FRET measurements, Chen’s group proposed a kinetic scheme for translocation, which included an initial powerstroke step followed by a thermal ratcheting step, thus providing insights into how the conformational plasticity of pseudoknots affects –1 PRF via selective modulation of late-stage translocation. Furthermore, their findings advance the current understanding of translocation and ribosome-induced mRNA unwinding (5).

Chen’s group also used several sm-FRET assays to directly characterize the effect of Onc112, an analogue of a proline-rich antimicrobial peptide (PrAMP) isolated from milkweed bug, on early elongation cycles during translation. They reported two different mechanisms of action of Onc112, including acceleration of the rejection rates of correct cognate tRNA during tRNA delivery and reduction of the percentage of active ribosomes to efficiently inhibit protein synthesis. Their findings provide further mechanistic insights into the mechanisms of action of PrAMPs and guidance to develop PrAMP-derived drugs (6).

In the future, Chen’s group intends to focus on developing single-molecule fluorescence tools with broad spatiotemporal detection windows. Thereafter, they will combine these newly developed tools with other approaches to capture dynamic ribosomal and CRISPR-Cas mechanisms and liquid-liquid phase separation.

References1. S. Peng et al., Angewandte Chemie 56, 6882–6885 (2017).2. S. Peng, W. Wang, C. Chen, Chem-Eur. J. 24, 1002–1009 (2018).3. S. Peng, W. Wang, C. Chen, J. Phys. Chem. B 122, 4844–4850 (2018).4. M. Yang et al., Cell Rep. 22, 372–382 (2018).5. B. Wu et al., Nucleic Acids Res. 46, 9736–9748 (2018).6. S. Peng et al., Protein Cell 9, 890–895 (2018).



The Research Group of Xianyang FangXianyang Fang

Xianyang Fang was born and raised in Hubei, China. He received his bachelor ’s degree in chemistry from Wuhan University in 2002, and his doctorate in biochemistry and molecular biology from the Institute of Biophysics, Chinese Academy of Sciences in 2008. In 2009, he joined the Structural Biophysics Laboratory, part of the National Cancer Institute at the U.S. National Institutes of Health campus in Frederick, Maryland, as a visiting postdoctoral researcher and later served as a research fellow in the laboratory of Yun-Xing Wang. During that time, he studied the structures of RNA, protein, and their complexes using small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy combined with computational modeling. In 2015, Fang was recruited to the School of Life Sciences, Tsinghua University, as a tenure-track assistant professor.

Fang’s research group consists of one postdoctoral fellow and 10 graduate students. The group integrates biochemical, biophysical, and molecular biological approaches to study the structures and molecular interactions of functional noncoding RNAs. The main research areas of the group are summarized below.

1. Uncovering the regulatory mechanisms of functional viral RNA elements

RNA viruses constitute a large number of human pathogens, such as dengue virus (DENV), Zika virus (ZIKV), hepatitis C virus, severe acute respiratory syndrome (SARS) coronavirus, HIV-1, and HTLV-1. These viruses have highly structured RNA genomes, which encode complex regulatory elements that play crucial roles in the viral life cycle, such as the modulation of viral transcription, translation, replication, packaging, and interactions with viral and host proteins. Increasing our knowledge of the viral RNA structure could lead to improvements in antiviral therapies. Therefore, Fang’s research group is focused on elucidating the high-resolution 3D structures and interactions of the regulatory RNAs found in HIV and other viral RNA genomes, and on understanding how such structures contribute to their functions in the viral life cycle.

2. Developing position-specific RNA labeling schemesStructural studies of RNA using traditional biophysical techniques,

including X-ray crystallography, NMR, and single-particle cryo-electron microscopy, are extremely challenging. SAXS and small-angle neutron scattering (SANS) has grown in popularity in recent years and could facilitate the study of the secondary and tertiary structures of RNAs in solution; however, the resolution is relatively low (1, 2). Thus, the research trend is toward integrative structural biology, a combination of structural data generated from the aforementioned structural techniques and distance data obtained from other biophysical and computational methods, which is highly valuable for the study of RNA structure and dynamics. Single-molecule fluorescence resonance energy transfer (sm-FRET), electron paramagnetic resonance (EPR), and X-ray scattering interferometry (XSI) can be used to obtain pairwise distance information, provided that the RNA molecules are site-specifically

labeled with fluorescent tags, spin labels, or gold nanoparticles, which is challenging. Fang’s research group is interested in developing fast and efficient RNA labeling schemes to enable implementation of such methods.

3. Characterizing the structure and function of large RNAs with integrative methods

Interest is growing in the field of long noncoding RNAs (lncRNAs). Fang’s research group is particularly interested in answering the following key questions: Do lncRNAs have well-defined 3D structures? What are the molecular determinants for the interaction between lncRNAs and their cofactors? How does the complex structure of lncRNAs determine their cellular functions? Although the group primarily uses biochemical approaches, such as selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) probing for secondary structural analysis, in combination with integrative methods such as SAXS/SANS, EPR, sm-FRET, and XSI for 3D structural analysis, they also combine these in vitro experiments with in vivo functional assays such as reverse genetics analysis.

In a recent study by Fang’s group (3), they investigated the 3D structures of a group of long non-coding subgenomic flavivirus RNAs (sfRNAs) using SHAPE probing, SAXS, and computational modeling (Figure 1). The study provided not only structural insight into the function of sfRNAs but also a general protocol for characterizing other lncRNAs by integrative structural biological approaches.

References1. X. Fang et al., Cell 155, 594–605 (2013).2. X. Fang et al., Curr. Opin. Struct. Biol. 30, 147–160 (2015).3. Y. Zhang et al., EMBO Rep., https://doi.org/10.15252/embr.201847016 (2019).


FIGURE 1. General protocol for 3D structural study of the long noncoding subgenomic f lavivirus RNA (sfRNA) from ZIKV by SAXS and computational modeling. This figure was modified from (3).

39CHAPTER 4

The Research Group of Jiawei WangJianwei Zeng, Jiawei Wang*

Jiawei Wang completed his bachelor ’s degree in 2001 at the Department of Materials Science & Engineering at the University of Science and Technology of China, and completed his doctorate in 2005 at the Institute of Physics of the Chinese Academy of Sciences. He then pursued his postdoctoral training between 2005 and 2008 in Zbigniew Dauter ’s laboratory at the Argonne National Laboratory near Chicago. In 2008, Wang joined the faculty of the School of Life Sciences at Tsinghua University.

Wang’s group comprises one staff member and seven graduate students. They have been developing computational methods to resolve complex problems in structural biology using X-ray crystallography and cryo-electron microscopy (cryo-EM), simultaneously elucidating the structural aspects of the bacterial phosphotransferase system. Thus far, Wang’s group has published more than 17 scientific articles in prestigious journals since 2015. The major contributions of Wang’s group are summarized below.

1. Structural analyses of bacterial phosphotransferase systemsThe bacterial phosphoenolpyruvate-dependent

phosphotransferase system (PTS) mediates carbohydrate uptake and phosphorylation. Wang’s group previously resolved the structures of membrane components from two PTS families (ascorbate and mannose). Ascorbate transporter UlaA exists in three distinct conformations (Figure 1), thus providing a complete overview of the transport cycle of ascorbate family members, suggesting an elevator-type, alternating-access mechanism (1, 2). The mannose-permease complex is different from the glucose and ascorbate families and exhibits three unrelated activities: (1) It mediates the uptake and phosphorylation of mannose and certain other hexoses, (2) permits bacteriophage lambda DNA injection across the inner membrane of Escherichia coli, and (3) serves as target receptors for class IIa, IId, and IIe bacteriocins. Wang’s group reported the structure of the transmembrane man-PTS complex derived from E. coli at a 3.52-Å resolution (3) (Figure 1), and identified the three distinct structural regions essential for recognizing class IIa and IId type bacteriocins, thus facilitating the development of bacteriocins for safe and efficient applications, such as their use in food preservatives and novel drugs to counter antibiotic-resistant pathogens.

2. Methodological development for structural biology analyses

Macromolecular crystallography and cryo-EM, complementary methods for molecular structure determination, can be combined to decipher

the structures of macromolecules (Figure 2) (4). Wang’s group developed an improved method for phasing crystal structures using cryo-EM data with no or limited noncrystallographic symmetry (5). The entire procedure iterates phase improvements through a combination of a reciprocal-space bias-removal method and a real-space fragment extraction method. Bijvoet differences associated with a certain amount of weak anomalous scattering of atoms on X-ray crystallography would yield additional benefits along with the cryo-EM phasing X-ray method, thus facilitating the determination of the correct absolute hand of the cryo-EM map (6).

De novo manual generation of atomic models for protein structures into the near-atomic resolution of the cryo-EM map would be imperative, but is time-consuming, tedious, and error-prone. Therefore, Wang’s group developed a program, EMBuilder, to automate model generation for cryo-EM maps (7).

The long-term objective of Wang’s group is to develop computational/experimental methods to resolve issues related to structural resolution during X-ray crystallography and cryo-EM.

References 1. P. Luo et al., Cell Discov. 4, 35 (2018).2. P. Luo et al., Nat. Struct. Mol. Biol. 22, 238–241 (2015).3. X. Liu et al., Cell Res. 29, 680–682 (2019).4. H. W. Wang, J. W. Wang, Protein Sci. 26, 32–39 (2017).5. J. Wang et al., Protein Cell 6, 919–923 (2015).6. J. Wang, J. Chai, H. Wang, FEBS J. 283, 1631–1635 (2016).7. N. Zhou, H. Wang, J. Wang, Sci. Rep. 7, 2664 (2017).

FIGURE 1. Topological and structural models of ascorbate and mannose PTS families .

FIGURE 2. A schematic representation of the protocol of combinatorial analysis using cryo-EM and X-ray crystallography. Reprinted from (5). CC BY (https://creativecommons.org/licenses/by/4.0/).

State�Key�Laboratory�of�Membrane�Biology,�Beijing�Advanced�Innovation�Center�for�Structural�Biology,�School�of�Life�Sciences,�Tsinghua�University,�Beijing,�China*Corresponding�author:�[email protected]


The Research Group of Ji j ie ChaiJizong Wang

Jijie Chai is an Alexander von Humboldt Professor at the University of Cologne, with his research group currently based at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, and an International Scholar at the Beijing Advanced Innovation Center for Structural Biology (ICSB). Prior to the position at the University of Cologne, Chai was a professor at Tsinghua University. He did his postdoctoral research on caspase activation and inhibition during apoptosis in Yigong Shi’s laboratory at Princeton University from 1999 to 2004. He was a recipient of the National Natural Science Award (Second Class) in 2017.

Chai’s research group at Tsinghua University consists of one associate investigator, four postdoctoral fellows, and nine graduate students, and focuses on structural and biochemical studies of plant receptor kinases (RKs) and nucleotide-binding (NB)-oligomerization-domain (NOD)-like receptors (NLRs). The research group has published more than 80 scientific articles in prestigious journals, including Science and Nature, and provided significant insight into the two large families of proteins. The major scientific

contributions made by Chai to the understanding of RKs and NLRs are summarized below.

1. Determination of conserved mechanism of RK activation but diverse modes of signaling complex assembly

Plant RKs are a large family of cell-surface receptors with a variable N-terminal extracellular domain (ECD), a transmembrane (TM) segment, and a conserved cytoplasmic kinase domain. Specific recognition of ligands via RK ECDs typically induces signaling associated with a plethora of physiological processes. Structural characterizations of a number of RKs by the Chai group revealed their ligand-recognition mechanisms and established ligand-induced homodimerization or heterodimerization as a conserved mechanism of RK activation (1–3).

Structures of the leucine-rich repeat (LRR) RK BRI1 (4), a receptor of the plant hormones brassinosteroids and FLS2 (5, 6) and an immune receptor of the N-terminal epitope of flagellin (flg22), showed that ligands act like “molecular glue” to combine receptors with the coreceptor BAK1 (Figure 1). This mechanism is conserved in the assembly of ligand-induced signaling complexes containing the LRR–RKs PEPR1 (7), PXY (8), or RGFR (9). These structural studies revealed the requirement of ligand-dependent heterodimerization of RKs for activation and led to the identification of receptors of root meristem growth factors (RGFR1–5) (9). Based on the mechanism of heterodimerization, the Chai group identified somatic embryogenesis receptor kinases (SERKs) as coreceptors of the peptide hormone phytosulfokine receptor (PSKR) (10). Interestingly, rather than acting as “molecular glue,” PSK allosterically induces

FIGURE 1. Assembly of homodimeric or heterodimeric RK-signaling complexes. Heterodimerization of RKs induced by ligands as “molecular glue” (top). PSK allosterically induces PSKR–SERK1 heterodimerization (middle, left) and SCR9 induces SRK9 homodimerization (middle, right). Ligand recognition by RK partnering with plasma membrane–localized proteins (bottom). All RK structures display their extracellular domains. The structures are shown with transparent surfaces and as cartoons, with Protein Data Bank codes indicated at the bottom of the structures.


41CHAPTER 5

the formation of a PSKR heterodimer with a SERK (10) (Figure 1). Additionally, the Chai group provided evidence of ligand-induced RK homodimerization required for activation and demonstrated chitin as being directly recognized by the lysin motif (LysM)-containing RK chitin elicitor receptor kinase 1 (CERK1) in Arabidopsis and triggering CERK1 homodimerization to initiate an immune response (11). This activation mode was further confirmed by the interaction of peptide SCR9 with its receptor S-receptor kinase 9 (SRK9) (12), which is important for self-incompatibility (Figure 1).

In addition to providing the structural basis of ligand recognition by single RKs (4–14), the Chai group revealed how an RK partners with a membrane-localized protein for recognition of its ligand(s). One example is the structures of the LRR ERECTA family (Erf ) RKs (15) involved in stomatal development. They showed that the LRR receptor-like protein TMM forms constitutive TMM–ERf complexes to recognize epidermal patterning factor (EPF)1 and EPF2. By contrast, single Erfs recognize EPFL4 and EPFL6, indicating that TMM functions as a specificity switch for recognition of different EPF peptides (Figure 1). Their more recent study (16) showed that direct recognition of rapid alkali nization factor (RALF) peptides by GPI-anchored proteins LLG1–3 promotes interaction of the rapid alkalinization factor with the malectin-domain-containing RK FER (Figure 1).

2. Autoinhibition, ligand recognition, and activation of animal NLRs

NLRs conserved in both animals and plants play crucial roles in innate immunity by acting as intracellular immune receptors (17). NLRs include a variable N-terminal domain, a conserved central NO D, and a C-terminal LRR domain. In 2013, the Chai group reported the crystal structure of inactive NLRC4 (18), revealing for the first time the autoinhibition mechanism of an NLR protein. They later reconstituted an NAIP2–NLRC4 inflammasome induced by the bacterial rod protein PrgJ (19). Cryo-electron microscopy (cryo-

EM) and biochemical data showed that the inflammasome forms wheel-like structures with a stoichiometry of 1:9 or 1:10 between NAIP2 and NLRC4 through NAIP2-induced self-activation of NLRC4 (19). A more recent study from the Chai group showed that bacterial flagellin binding stabilizes the active conformation of NAIP5 (20) to allow the self-activation of NLRC4. Collectively, structural studies by the Chai group revealed the mechanisms of autoinhibition, ligand recognition, and activation of NLRC4 and NAIP (Figure 2), laying a foundation for the study of the mechanisms of action of other NLRs.

3. Plant NLR resistosomeNLRs are narrowly referred to as plant-disease-resistance (R)

proteins and directly or indirectly mediate immune sensing of pathogen-effector proteins. The most direct evidence for an indirect-recognition model comes from structural studies of Pto complexed with the effector protein AvrPto (21) or AvrPtoB (22, 23) to trigger an immune response through the coiled-coil (CC)-NLR Prf. Structural data revealed that AvrPto inhibits FLS2-mediated immunity (24) and provided a foundation for the decoy model of plant NLR recognition of ligands (25).

Two recent studies (26, 27) by the Chai group offered unprecedented insights into the mechanisms of action of plant NLRs. Cryo-EM structures revealed the mechanisms of CC-NLR ZAR1 autoinhibition and ZAR1–RKS1 recognition of the effector AvrAC-uridylated PBL2 (26). Importantly, they reconstituted an active ZAR1–RKS1–PBL2 (uridylated form) complex in vitro (referred to as the ZAR1 resistosome), revealing its formation of a wheel-like pentamer (27). Furthermore, they showed that the first α-helix in the CC domain buried in the inactive ZAR1 structure forms a funnel-shaped structure at the N-terminus of the ZAR1 resistosome that is required for ZAR1 integration into the plasma membrane and initiation of ZAR1-mediated cell death and plant-disease resistance (Figure 3). These data led them to hypothesize that the ZAR1 resistosome functions as a channel or pore to perturb

FIGURE 2. Activation of the NAIP5–NLRC4 inflammasome in mice. Experimentally determined structures are indicated by their Protein Data Bank codes or EMDB ID. NTD, N-terminal domain; ID, integrated domain; BIR, baculoviral inhibition of apoptosis protein repeat; FLiC_D0L, a flagellin derivative with its N- and C-terminal regions fused together. Refer to the text for definitions of other acronyms.


the stability of the plasma membrane in order to promote the initiation of downstream signaling. Although this model requires further validation, these two studies provided clues regarding the biochemical functions of CC-NLRs and have important implications for plant cell death and immunity. Another finding was that fold switching occurs in ZAR1 during activation, as the structure of the inactive CC of ZAR1 resembles that of Rx (28) but is strikingly different from that of MLA10 (29).

4. The nucleotide-switch mechanism of NLRsNLR proteins are widely known as an ADP–ATP switch, with

ADP- and ATP-bound forms representing inactive and active states, respectively. However, the underlying mechanism associated with the switch activity remained elusive until a recent study (26) captured the primed state of ZAR1 within the structure of the ZAR1–RKS1–PBL2UMP complex, which showed that PBL2UMP binding to RKS1 allosterically induces conformational changes in ZAR1NBD to attenuate ZAR1 binding of ADP. ATP or dATP binding by primed ZAR1 further triggers structural reorganization of the NOD to form the ZAR1 resistosome (Figure 3). These results indicate that conformational changes induced by both ligand (AvrAC-uridylated PBL2) and d/ATP are required for full activation of ZAR1.

The long-term goals of the Chai group are to understand the structural mechanisms of action of diverse NLRs and engineer plant NLRs with different specificity for agricultural practice.

References1. Z. Han, Y. Sun, J. Chai, Curr. Opin. Plant Biol. 20, 55–63 (2014).

2. H. Zhang et al., Mol. Plant. 9, 1454–1463 (2016).3. W. Song et al., Curr. Opin. Struct. Biol. 43, 18–27 (2017).4. J. She et al., Nature 474, 472–476 (2011).5. Y. Sun et al., Cell Res. 23, 1326–1329 (2013).6. Y. Sun et al., Science 342, 624–628 (2013).7. J. Tang et al., Cell Res. 25, 110–120 (2015).8. H. Zhang et al., Mol. Plant 9, 1406–1414 (2016).9. W. Song et al., Cell Res. 26, 674–685 (2016).10. J. Wang et al., Nature 525, 265–268 (2015).11. T. Liu et al., Science 336, 1160–1164 (2012).12. R. Ma et al., Cell Res. 26, 1320–1329 (2016).13. H. Zhang et al., Cell Res. 26, 543–555 (2016).14. X. Zhang et al., Nat. Commun. 8, 1331 (2017).15. G. Lin et al., Genes Dev. 31, 927–938 (2017).16. Y. Xiao et al., Nature 572, 270–274 (2019).17. Z. Hu, J. Chai, Curr. Top. Microbiol. Immunol. 397, 23–42 (2016).18. Z. Hu et al., Science 341, 172–175 (2013).19. Z. Hu et al., Science 350, 399–404 (2015).20. X. Yang et al., Cell Res. 28, 35–47 (2018).21. W. Xing et al., Nature 449, 243–247 (2007).22. J. Dong et al., Plant Cell. 21, 1846–1859 (2009).23. W. Cheng et al., Cell Host Microbe 10, 616–626 (2011).24. T. Xiang et al., Curr. Biol. 18, 74–80 (2008).25. J. M. Zhou, J. Chai, Curr. Opin. Microbiol. 11, 179–185 (2008).26. J. Wang et al., Science 364, eaav5868 (2019a).27. J. Wang et al., Science 364, eaav5870 (2019b).28. W. Hao et al., J. Biol. Chem. 288, 35868–35876 (2013).29. T. Maekawa et al., Cell Host Microbe 9, 187–199 (2011).

FIGURE 3. Activation of the ZAR1 resistosome from Arabidopsis . Experimentally determined structures are indicated by their Protein Data Bank codes. Color codes are indicated.

43CHAPTER 5

The Research Group of Sen-Fang SuiShan Sun

Sen-Fang Sui obtained both his bachelor ’s degree in precision instruments and mechanology (1970) and his Master ’s degree in solid-state physics (1981) from Tsinghua University. He earned his doctoral degree in biophysics in 1988 from the Technical University of Munich in Germany. Sui started his own research group as an associate professor in the Department of Biological Sciences and Biotechnology—renamed the “School of Life Sciences” in 2009—at Tsinghua University in 1989 and was promoted to full professor in 1991. He was further appointed as dean of the Department of Biological Sciences and Technology of Tsinghua University in 1992 and held this position until 1995.

Sui is a member of the Chinese Academy of Sciences and has received numerous awards, including the 1989 Outstanding Young Teacher Award; the 2001 First Prize of the Science and Technology Award of Chinese Universities (Natural Science); the 2005 Second Prize of National Natural Science Award; the 2010 Qian Lin-Zhao Award from the Chinese Electron Microscopy Society; the 2015 Special Contribution Award of the School of Life Sciences, Tsinghua University; and the 2017 Lifetime Achievement Award from the Cryo-Electron Microscopy (Cryo-EM) Subgroup of the Biophysical Society of China. Sui currently serves as the vice president of the Chinese Society of Biochemistry and Molecular Biology, is a scientific advisory board member of China’s State Key Laboratory of Membrane Biology, and is an editorial board member of the Journal of Structural Biology.

Since 1989, when he first set up his own laboratory, Sui’s research has focused on applying three-dimensional (3D) EM to biology. In the early 1990s, his laboratory focused on two-dimensional (2D) electron crystallography, leading to the development of a novel method for 2D crystallization of proteins on a lipid layer. In the latter half of the 1990s, Sui’s attention shifted to the use of the single-particle reconstruction technique. At present, Sui’s laboratory uses this technique in combination with other biochemical, biophysical, and cellular methods to answer long-standing and emerging questions involving the structures, functions, and mechanisms of gigantic protein complexes, macromolecular machines, and membrane proteins, and to elucidate the molecular mechanisms of membrane trafficking. His group currently consists of one associate professor, four staff members, five postdoctoral fellows, and 11 graduate students. To date, Sui’s research group has published more than 150 scientific articles in prestigious journals such as Nature, Science, Nature Structural Molecular Biology, Science Advances, Nature Communications, Nature Plants, PNAS, and Cell Research. The major contributions of Sui’s research group are summarized below.

1. Resolving the structures of gigantic protein complexesPhotosynthesis is a process by which photosynthetic organisms

use solar energy to convert water and carbon dioxide into oxygen and various organic compounds that form the basis of almost all life on Earth. To make full use of energy from sunlight, organisms have developed various light-harvesting systems to collect and transfer

sunlight to the core complex of two photosystems: photosystem I (PSI) and photosystem II (PSII) (in which photosynthesis takes place). The phycobilisome (PBS) complex is the largest known light-harvesting complex and part of the primary light-harvesting antenna complex in cyanobacteria and red algae. Since its discovery more than 50 years ago, extensive efforts have been made to understand how this huge supramolecular complex is assembled and how energy is transferred within its structure.

Sui’s group initially resolved the structures of an intact PBS from a cyanobacterium and its association with PSII at medium resolutions using negative-stained EM (1). Subsequently, using single-particle cryo-electron microscopy, they resolved the first 3D structure of an intact PBS from a red alga at an atomic resolution of 3.5 Å (Figure 1, left panel) (2). This complex was confirmed to be incredibly large, possessing a molecular mass of roughly 18.0 megadaltons and consisting of 862 protein subunits and 2,048 chromophores,

FIGURE 1. Overall structure of the PBS from the red alga G. pacifica shown as a surface representation (left), and the skeleton formed by the linker proteins shown as a cartoon representation (right).

FIGURE 2. Structure of the α-SNAP–SNARE subcomplex . (A) EM density map of the α-SNAP–SNARE subcomplex colored according to the local resolution; the right image shows a section view of the interior of the map. (B) Cryo-EM density maps of the whole SNARE complex (left) and representative layers of the SNARE complex superimposed with respective atomic stick models (right). Adapted from (8). CC BY-NC (https://creativecommons.org/licenses/by-nc/4.0/).



in which a number of novel protein structures were identified for the first time. Visualization of the overall structure at such a high resolution further helped elucidate both the mechanisms underlying specific interactions between phycobiliproteins and linkers as well as the skeleton formed by linkers for the assembly of the intact PBS (Figure 1, right panel). This work provided a strong structural basis for understanding the assembly of such a gigantic complex and the mechanisms of energy transfer within it. Sui’s group has resolved the high-resolution structures of the PSII–light-harvesting antenna supercomplex of a diatom (3), in addition to the PSI–light-harvesting antenna supercomplexes of both a green alga (4) and a red alga (5). Collectively, the structures of these gigantic protein complexes provide an important foundation for elucidating the energy transfer and dissipation pathways of various photosystem-antenna systems and the evolution of oxygenic photosystems as a whole across species.

2. Revealing the molecular mechanisms of membrane traffickingProper functioning of cells depends on the successful

transportation of the proper molecules to the proper place at the appropriate time. The primary mode of molecule transportation in cells is vesicle-mediated membrane trafficking, which depends upon the continual fission and fusion of cell membranes. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is the core machinery for membrane fusion and is a highly stable four-helix bundle. After membrane fusion, the SNARE complex binds to N-ethylmaleimide-sensitive factors (NSFs) and soluble NSF attachment proteins (SNAPs) to form a transient 20S complex, in which NSF and SNAP coordinate to disassemble the SNARE complex into individual proteins available for recycling. Prior to Sui’s work on this process, the detailed mechanisms behind SNARE complex disassembly remained obscure.

Sui’s group resolved the negative-stain EM (6) and cryo-EM structures of the entire 20S complex (7, 8). Moreover, they developed a novel image-processing method to overcome an intrinsic flexibility issue within the 20S complex and resolved the structure of the α-SNAP–SNARE subcomplex at an overall resolution of 7.35 Å (7), which has recently been improved to a resolution of 3.9 Å (Figure 2) (8). The resolved structure indicated that four α-SNAP molecules form a right-handed cylindrical assembly that wraps around the left-handed SNARE helical bundle within the 20S complex, with comprehensive contact occurring between the SNAPs and SNARE proteins. Further electrophysiological and biochemical data demonstrated that α-SNAPs mainly act on two positions of the vesicle-associated membrane protein (VAMP)—one of the four SNARE proteins—for disassembly. Moreover, they defined the contact between the amino terminus of the SNARE bundle and the pore loop of the NSF-D1 domain, which also plays a critical role in the disassembly of the SNARE complex. Based on these findings, they proposed a rotation model of α-SNAP-mediated SNARE complex disassembly.

Vesicle budding and fission from the donor compartment is the first step of membrane trafficking. Membrane fission involving

interior budding (i.e., toward the cytosol) is well characterized. Conversely, the mechanisms underlying membrane budding and fission in the opposite direction (i.e., away from the cytosol) are poorly understood. Such “inverse” membrane dynamics are facilitated by the endosomal sorting complex required for transport (ESCRT)-III polymers and the vacuolar protein sorting 4 (Vps4) protein. Vps4 oligomerizes into a functional hexamer upon binding to ATP, resulting in elevated ATPase activity. The Vps4 hexamer—facilitated by its cofactor protein, Vps twenty-associated-1 (Vta1)—uses the energy from ATP hydrolysis to disassemble the ESCRT-III complex into its constituent proteins.

Using single-particle cryo-EM, Sui’s group resolved the structures of the ATP-bound Vps4 hexamer and its complex with Vta1 at resolutions of 3.9 Å and 4.2 Å, respectively (Figure 3) (9). In both structures, six Vps4 subunits are arranged into a spiral-shaped ring, and the Vta1 dimers are located at the periphery of the ring connecting two adjacent Vps4 subunits through two different interaction modes, facilitating the formation of the Vps4 hexamer. Further investigation using a single-molecule fluorescence approach suggested that Vta1 acts as an assembling factor to aid the formation of the active Vps4 hexamers associated with the ESCRT-III filaments and likewise promotes the function of Vps4. These findings not only explained the assembly of the ATP-bound Vps4 hexamer in the absence and presence of Vta1, but further elucidated the mechanisms underlying the ATP-dependent oligomerization of Vps4 and its elevated activity in complex with Vta1.

References1. L. Chang et al., Cell Res. 25, 726–737 (2015).2. J. Zhang et al., Nature 551, 57–63 (2017).3. X. Pi et al., Science 365, eaax4406 (2019).4. X. Qin et al., Nat. Plants 5, 263–272 (2019).5. X. Pi et al., Proc. Natl. Acad. Sci. U. S. A. 115, 4423–4428 (2018).6. L. F. Chang et al., Nat. Struct. Mol. Biol. 19, 268–275 (2012).7. Q. Zhou et al., Cell Res. 25, 551–560 (2015).8. X. Huang et al., Sci. Adv. 5, eaau8164 (2019).9. S. Sun et al., Nat. Commun. 8, 16064 (2017).

FIGURE 3. Structures of the Vps4 hexamer in the ATP-bound state (left) and its complex with Vta1 cofactors (right). Each protein subunit is represented in a different color. Vta1 subunits are shown as cylinders.

Center for International Cooperation and ExchangeSince its establishment in 2015, the center, located in the Medical Science Building at Tsinghua University, has attached great importance to cultivating international collaboration and the exchange of ideas. It holds international academic conferences in various fields twice a year and has attracted more than 100 renowned professors, including Nobel laureates, to provide principle investigators and young students with cutting-edge information about scientific research. A few of the conferences held in recent years are summarized below.

2016 International Conference on Spliceosome Kinetics, Catalysis, and Regulation

Many outstanding scientists in the field of spliceosome and RNA research presented, including Joan Steitz of Yale University and Christine Guthrie of the University of California, San Francisco, who discovered the core component of the spliceosome; Reinhard Lührmann of the Max Planck Institute for Biophysical Chemistry and Yigong Shi of Tsinghua University, who conducted pioneering research on the structure of the spliceosome; and Thomas Steitz, who won the 2009 Nobel Prize in Chemistry for the study of ribosome structure and function.

2017 International Conference on Structural Biology

The theme of the conference was “Biological Macromolecules: Structure, Catalysis, and Regulation,” and included five topics: membrane proteins and molecular machines; epigenetics; gene expression and regulation; spliceosome: assembly, transition, and catalysis; and new advancements in nuclear magnetic resonance (NMR) technology. Attending scientists included international scholars such as Kurt Wüthrich, 2002 Nobel Prize laureate in Chemistry from the Scripps Research Institute, ETH Zurich, and the iHuman Institute of ShanghaiTech University; Brian Kobilka, 2012 Nobel Prize laureate in Chemistry from Stanford University; Robert Roeder, 2003 Lasker Award laureate from Rockefeller University; and Dinshaw Patel, Member of the U.S. National Academy of Sciences (NAS) from the Sloan Kettering Institute.

2018 International Symposium on Computational Structural Biology and Biophysics

This symposium covered four major topics: computational methods for cryo-electron microscopy structure determination; computations in X-ray, NMR, and small-angle X-ray scattering technology; protein structure analysis, prediction, and design; and 3D genome and RNA structrome: computation and modeling. In all, 22 outstanding scientists were invited, including Michael Levitt from Stanford University (2013 Nobel Prize laureate in Chemistry); Chris Sander from Harvard Medical School (Member of the NAS); and Barry Honig from Columbia University (Member of the NAS).

2019 International Conference on Neurostructural Biology

Topics covered at this conference included brain connectomics and advanced brain imaging technologies, structural studies of neural molecules, and phase separation and the neuronal cytoskeleton. Twenty-six world-renowned structural biologists and neurobiologists attended, including Jeff Lichtman (Harvard University), Steven McKnight (University of Texas Southwestern Medical Center), Yuh Nung Jan (University of California, San Francisco), and Mingjie Zhang (Hong Kong University of Science and Technology).

Advanced Innovation Lecture Series for Structural Biology

The center invites many preeminent experts as part of an ongoing lecture series. Speakers have included Wei Yang (U.S. National Institutes of Health); Robert Glaeser (University of California, Berkeley and NAS); Eva Nogales (Lawrence Berkeley National Laboratory and University of California, Berkeley); John Kuriyan (University of California, Berkeley); Michael Rosen (University of Texas Southwestern Medical Center); Anna Marie Pyle (Yale University); Michael Levitt (Stanford University); Clare Waterman (U.S. National Institutes of Health); Stephen Young (University of California, Los Angeles); Jay Horton (University of Texas Southwestern Medical Center); Robert Langer (Massachusetts Institute of Technology); Caroline Dean (John Innes Centre, University of York); Arnold Levine (Institute for Advanced Study, Princeton); Joachim Frank (Nobel Laureate, Columbia University); Wolfgang Baumeister (Max Planck Institute of Biochemistry); and Angela Gronenborn (University of Pittsburgh). This lecture series is notable in providing teachers and students the opportunity to closely interact with renowned academic scholars.

2016 International Conference on Spliceosome Kinetics, Catalysis and Regulation

2019 International Conference on Neurostructural Biology


www.icsb.tsinghua.edu.cnTsinghua University

Haidian DistrictBeijing 100084, China

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