ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of HertfordshireHatfield, Hertfordshire, UK

For further volumes:http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols andmethodologies in the critically acclaimedMethods in Molecular Biology series. The series wasthe first to introduce the step-by-step protocols approach that has become the standard in allbiomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagentsneeded to complete the experiment, and followed by a detailed procedure that is supportedwith a helpful notes section offering tips and tricks of the trade as well as troubleshootingadvice. These hallmark features were introduced by series editor Dr. John Walker andconstitute the key ingredient in each and every volume of the Methods in Molecular Biologyseries. Tested and trusted, comprehensive and reliable, all protocols from the series areindexed in PubMed.

Intrinsically Disordered Proteins

Methods and Protocols

Edited by

Birthe B. Kragelund

Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, København N,Denmark

Karen Skriver

Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen,Copenhagen, Denmark

EditorsBirthe B. KragelundStructural Biology and NMRLaboratoryDepartment of BiologyUniversity of CopenhagenKøbenhavn N, Denmark

Karen SkriverLinderstrøm-Lang Centre for Protein ScienceDepartment of BiologyUniversity of CopenhagenCopenhagen, Denmark

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-0716-0523-3 ISBN 978-1-0716-0524-0 (eBook)https://doi.org/10.1007/978-1-0716-0524-0

© Springer Science+Business Media, LLC, part of Springer Nature 2020Chapter 24 is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter.This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,expressed or implied, with respect to the material contained herein or for any errors or omissions that may have beenmade. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface

Intrinsically disordered proteins (IDPs) challenge current views on biomolecular communi-cation because these proteins participate in functional biological complexes despite their lackof 3D structures, a scenario which until recently was unimaginable. An important key tounlocking the enigmatic nature of IDPs is the development of appropriate experimentalmethods and tools for their analysis, and derivation of mechanistic models. Due to theirdynamic nature, IDPs expand the types of possible complexes, and they are highly challeng-ing to investigate with traditional methods. However, with a remarkable rate, novel modesof operations are being uncovered, leading to new methods, tools, and strategies developedand modified specifically for studies of IDPs. Therefore, aMethods in Molecular Biology bookon Intrinsically Disordered Proteins: Methods and Protocols is warranted. This edition follows,but is independent from, the first successful books on the topic in this series publishedalmost 10 years ago. The edition covers methods to study IDPs in general and includesmethods to study very recent topics such as extremely high-affinity disordered complexes,kinetics that evades established concepts, liquid–liquid phase separation, and novel disorder-driven allosteric mechanisms. Written in the highly successful Methods in Molecular Biol-ogy™ series format, the chapters include the kind of detailed description and implementa-tion advice that is crucial for dissemination of good practice and for getting optimal andreliable results in the laboratory. Thorough and intuitive Intrinsically Disordered Proteins:Methods and Protocols helps scientists with different backgrounds to further their investiga-tions of these fascinating and dynamic molecules.

Copenhagen, Denmark Karen SkriverKøbenhavn, Denmark Birthe B. Kragelund

v

Acknowledgements

First and foremost, we owe a big thank you to all of themore than one hundred authors who,with impressive engagement, have contributed with a specific chapter to this special issue onIntrinsically Disordered Proteins (IDPs). We have been overwhelmed and impressed by thequality of the chapters, the many important details of hands-on tips and tricks, and theproudness of presenting a useful protocol. We have met a true excitement, which all togetherunderscore the eagerness and readiness to share important methods and protocols charac-teristic of the IDP field. We owe a special thank you to those of you who stepped in at the lastminute.

Thank you all!Second, we are grateful to all our students and post docs who have taken the time and

effort to go through the protocols with a user’s eye, and for your many suggestions toimprove the usefulness of the protocols. Lasse, Pernille, Inna, Sarah, Edoardo, Katrine,Christian, Frederik, Maria—thank you!

Finally, we acknowledge the support from John Walker and Patrick Marton fromSpringer for valuable feedback during the process and the support of the REPIN Centrefunded by the Novo Nordisk Foundation, enabling us to allocate the needed time to get itall together.

Karen and Birthe

vii

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

PART I SEQUENCE PROPERTIES

1 Disorder for Dummies: Functional Mutagenesis of TransientHelical Segments in Disordered Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Gary W. Daughdrill

2 Computational Prediction of Intrinsic Disorder in Protein Sequenceswith the disCoP Meta-predictor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Christopher J. Oldfield, Xiao Fan, Chen Wang, A. Keith Dunker,and Lukasz Kurgan

3 Computational Prediction of Disordered Protein Motifs Using SLiMSuite . . . . . 37Richard J. Edwards, Kirsti Paulsen, Carla M. Aguilar Gomez,and Asa Perez-Bercoff

4 How to Annotate and Submit a Short Linear Motif to the EukaryoticLinear Motif Resource . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Marc Gouw, Jesus Alvarado-Valverde, Jelena Calyseva, Francesca Diella,Manjeet Kumar, Sushama Michael, Kim Van Roey, Holger Dinkel,and Toby J. Gibson

5 Analyzing the Sequences of Intrinsically Disordered Regionswith CIDER and localCIDER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103Garrett M. Ginell and Alex S. Holehouse

6 Exploring Protein Intrinsic Disorder with MobiDB . . . . . . . . . . . . . . . . . . . . . . . . . 127Alexander Miguel Monzon, Andras Hatos, Marco Necci, Damiano Piovesan,and Silvio C. E. Tosatto

PART II EVOLUTION

7 An Easy Protocol for Evolutionary Analysis of IntrinsicallyDisordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Janelle Nunez-Castilla and Jessica Siltberg-Liberles

PART III PRODUCTION

8 Expression and Purification of an Intrinsically Disordered Protein. . . . . . . . . . . . . 181Karamjeet K. Singh and Steffen P. Graether

9 Production of Intrinsically Disordered Proteins for Biophysical Studies:Tips and Tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195Christian Parsbæk Pedersen, Pernille Seiffert, Inna Brakti,and Katrine Bugge

ix

10 Recombinant Production of Monomeric Isotope-EnrichedAggregation-Prone Peptides: Polyglutamine Tracts and Beyond . . . . . . . . . . . . . . 211Albert Escobedo, Giulio Chiesa, and Xavier Salvatella

11 Cell-Free Protein Synthesis of Small Intrinsically DisorderedProteins for NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233Linnea Isaksson and Anders Pedersen

PART IV DYNAMICS, ENSEMBLES, AND STRUCTURES

12 Structural Analyses of Intrinsically Disordered Proteins by Small-AngleX-Ray Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249Amin Sagar, Dmitri Svergun, and Pau Bernad�o

13 Determining Rg of IDPs from SAXS Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271Ellen Rieloff and Marie Skepo

14 Obtaining Hydrodynamic Radii of Intrinsically DisorderedProtein Ensembles by Pulsed Field Gradient NMR Measurements . . . . . . . . . . . . 285Sarah Leeb and Jens Danielsson

15 Quantitative Protein Disorder Assessment Using NMR Chemical Shifts . . . . . . . 303Jakob T. Nielsen and Frans A. A. Mulder

16 Determination of pKa Values in Intrinsically Disordered Proteins . . . . . . . . . . . . . 319Brandon Payliss and Anthony Mittermaier

17 Paris-DECOR: A Protocol for the Determination of FastProtein Backbone Amide Hydrogen Exchange Rates . . . . . . . . . . . . . . . . . . . . . . . . 337Rupashree Dass and Frans A. A. Mulder

PART V ENSEMBLES BY COMPUTATION

18 Predicting Conformational Properties of IntrinsicallyDisordered Proteins from Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347Kiersten M. Ruff

19 Enhanced Molecular Dynamics Simulations of IntrinsicallyDisordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391Matteo Masetti, Mattia Bernetti, and Andrea Cavalli

20 Computational Protocol for Determining ConformationalEnsembles of Intrinsically Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413Robert B. Best

21 Computing, Analyzing, and Comparing the Radius of Gyrationand Hydrodynamic Radius in Conformational Ensemblesof Intrinsically Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429Mustapha Carab Ahmed, Ramon Crehuet, and Kresten Lindorff-Larsen

PART VI DETERMINANTS OF INTERACTIONS

22 Binding Thermodynamics to Intrinsically Disordered Protein Domains. . . . . . . . 449Arne Schon and Ernesto Freire

x Contents

23 Analysis of Multivalent IDP Interactions: Stoichiometry,Affinity, and Local Concentration Effect Measurements . . . . . . . . . . . . . . . . . . . . . 463Samuel Sparks, Ryo Hayama, Michael P. Rout, and David Cowburn

24 NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions . . . . . . 477Christopher A. Waudby and John Christodoulou

25 Measuring Effective Concentrations Enforced by IntrinsicallyDisordered Linkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505Charlotte S. Sørensen and Magnus Kjaergaard

26 Determining the Protective Activity of IDPs Under PartialDehydration and Freeze-Thaw Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519David F. Rend�on-Luna, Paulette S. Romero-Perez,Cesar L. Cuevas-Velazquez, Jose L. Reyes,and Alejandra A. Covarrubias

27 Screening Intrinsically Disordered Regions for Short LinearBinding Motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529Muhammad Ali, Leandro Simonetti, and Ylva Ivarsson

PART VII INTERACTIONS ON SURFACES

28 Probing IDP Interactions with Membranes by FluorescenceSpectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555Diana Acosta, Tapojyoti Das, and David Eliezer

29 Protocol for Investigating the Interactions Between IntrinsicallyDisordered Proteins and Membranes by Neutron Reflectometry. . . . . . . . . . . . . . 569Alessandra Luchini and Lise Arleth

30 Interactions of IDPs with Membranes Using Dark-State ExchangeNMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585Tapojyoti Das, Diana Acosta, and David Eliezer

PART VIII BINDING KINETICS AND MECHANISMS

31 Determination of Binding Kinetics of Intrinsically DisorderedProteins by Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611Julie M. Leth and Michael Ploug

32 Measuring and Analyzing Binding Kinetics of Coupled Foldingand Binding Reactions Under Pseudo-First-Order Conditions . . . . . . . . . . . . . . . 629Kristine Steen Jensen

33 Understanding Binding-Induced Folding by Temperature Jump. . . . . . . . . . . . . . 651Angelo Toto, Francesca Troilo, Francesca Malagrino,and Stefano Gianni

34 Determining Binding Kinetics of Intrinsically DisorderedProteins by NMR Spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663Ke Yang, Munehito Arai, and Peter E. Wright

Contents xi

PART IX HIGHER ORDER-PHASE SEPARATION AND FIBRILLATION

35 Determination of Protein Phase Diagrams by Centrifugation. . . . . . . . . . . . . . . . . 685Nicole M. Milkovic and Tanja Mittag

36 In Vitro Transition Temperature Measurement of Phase-SeparatingProteins by Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703Jack Holland, Michael D. Crabtree, and Timothy J. Nott

37 Walking Along a Protein Phase Diagram to Determine CoexistencePoints by Static Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715Ivan Peran, Erik W. Martin, and Tanja Mittag

38 Expression and Purification of Intrinsically Disordered Aβ Peptideand Setup of Reproducible Aggregation Kinetics Experiment . . . . . . . . . . . . . . . . 731Sara Linse

39 Measuring Interactions Between Tau and Aggregation Inducerswith Single-Molecule Forster Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . 755Sanjula P. Wickramasinghe and Elizabeth Rhoades

PART X MODIFICATION AND TARGETING

40 Detection of Multisite Phosphorylation of IntrinsicallyDisordered Proteins Using Phos-tag SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . 779Mihkel Ord and Mart Loog

41 Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR . . . . . . . . . . 793Manon Julien, Chafiaa Bouguechtouli, Ania Alik, Rania Ghouil,Sophie Zinn-Justin, and Francois-Xavier Theillet

42 Detection of Multisite Phosphorylation of Intrinsically DisorderedProteins Using Quantitative Mass-Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819Ervin Valk, Artemi Maljavin, and Mart Loog

43 Targeting an Intrinsically Disordered Protein by Covalent Modification . . . . . . . 835Hung Huy Nguyen, Peter Abranyi-Balogh, Laszl�o Petri, Attila Meszaros,Kris Pauwels, Guy Vandenbussche, Gyorgy Mikl�os Keseru, and Peter Tompa

PART XI IN CELL AND INTERACTOMES

44 Recording In-Cell NMR-Spectra in Living Mammalian Cells . . . . . . . . . . . . . . . . . 857Irena Matecko-Burmann and Bjorn M. Burmann

45 In-Cell NMR of Intrinsically Disordered Proteins in Mammalian Cells . . . . . . . . 873Juan A. Gerez, Natalia C. Prymaczok, and Roland Riek

46 Analyzing IDPs in Interactomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895Vladimir N. Uversky

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947

xii Contents

Contributors

PETER ABRANYI-BALOGH • Medicinal Chemistry Research Group, Research Centre forNatural Sciences, Hungarian Academy of Sciences, Budapest, Hungary

DIANA ACOSTA • Department of Biochemistry, Weill Cornell Medical College of CornellUniversity, New York, NY, USA; Brain and Mind Research Institute, Weill CornellMedical College of Cornell University, New York, NY, USA

CARLA M. AGUILAR GOMEZ • School of Biotechnology and Biomolecular Sciences, University ofNew South Wales, Sydney, NSW, Australia

MUSTAPHA CARAB AHMED • Department of Biology, Structural Biology and NMRLaboratory, Linderstrøm-Lang Centre for Protein Science, University of Copenhagen,Copenhagen N, Denmark

MUHAMMAD ALI • Department of Chemistry, BMC, Uppsala, SwedenANIA ALIK • Universite Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the

Cell (I2BC), Gif-sur-Yvette, FranceJESUS ALVARADO-VALVERDE • Structural and Computational Biology Unit, European

Molecular Biology Laboratory, Heidelberg, Germany; Faculty of Biosciences, Collaborationfor Joint PhD Degree Between EMBL and Heidelberg University, Heidelberg, Germany

MUNEHITO ARAI • Department of Life Sciences, Graduate School of Arts and Sciences, TheUniversity of Tokyo, Tokyo, Japan

LISE ARLETH • Structural Biophysics, X-ray and Neutron Science, Niels Bohr Institute,University of Copenhagen, Copenhagen, Denmark

PAU BERNADO • Centre de Biochimie Structurale, INSERM, CNRS, Universite deMontpellier, Montpellier, France

MATTIA BERNETTI • Scuola Internazionale Superiore di Studi Avanzati, Trieste, ItalyROBERT B. BEST • Laboratory of Chemical Physics, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USACHAFIAA BOUGUECHTOULI • Universite Paris-Saclay, CEA, CNRS, Institute for Integrative

Biology of the Cell (I2BC), Gif-sur-Yvette, FranceINNA BRAKTI • Department of Biology, Structural Biology and NMR Laboratory and the

Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, Copenhagen N,Denmark

KATRINE BUGGE • Department of Biology, Structural Biology and NMR Laboratory and theLinderstrøm-Lang Centre for Protein Science, University of Copenhagen, Copenhagen N,Denmark

BJORN M. BURMANN • Wallenberg Centre for Molecular and Translational Medicine,University of Gothenburg, Goteborg, Sweden; Department of Chemistry and MolecularBiology, University of Gothenburg, Goteborg, Sweden

JELENA CALYSEVA • Structural and Computational Biology Unit, European MolecularBiology Laboratory, Heidelberg, Germany; Faculty of Biosciences, Collaboration for JointPhD Degree Between EMBL and Heidelberg University, Heidelberg, Germany

ANDREA CAVALLI • Department of Pharmacy and Biotechnology, Alma Mater Studiorum—Universita di Bologna, Bologna, Italy; Computational and Chemical Biology, IstitutoItaliano di Tecnologia, Genoa, Italy

xiii

GIULIO CHIESA • Institute for Research in Biomedicine (IRB Barcelona), The BarcelonaInstitute of Science and Technology, Barcelona, Spain; Joint BSC-IRBResearch Programmein Computational Biology, Barcelona, Spain; Department of Biomedical Engineering andBiological Design Center, Boston University, Boston, MA, USA

JOHN CHRISTODOULOU • Institute of Structural and Molecular Biology, UCL and BirkbeckCollege, London, UK

ALEJANDRA A. COVARRUBIAS • Departamento de Biologıa Molecular de Plantas, Instituto deBiotecnologıa, Universidad Nacional Aut�onoma de Mexico, Cuernavaca, Morelos, Mexico

DAVID COWBURN • Department of Biochemistry, Albert Einstein College of Medicine, Bronx,NY, USA

MICHAEL D. CRABTREE • Department of Biochemistry, University of Oxford, Oxford, UKRAMON CREHUET • Department of Biology, Structural Biology and NMR Laboratory,

Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, Copenhagen N,Denmark; Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona,Spain

CESAR L. CUEVAS-VELAZQUEZ • Departamento de Biologıa Molecular de Plantas, Instituto deBiotecnologıa, Universidad Nacional Aut�onoma de Mexico, Cuernavaca, Morelos, Mexico;Departamento de Bioquımica, Facultad de Quımica, Universidad Nacional Aut�onoma deMexico, Mexico City, Mexico

JENS DANIELSSON • Department of Biochemistry and Biophysics, Arrhenius Laboratories ofNatural Sciences, Stockholm University, Stockholm, Sweden

TAPOJYOTI DAS • Department of Biochemistry, Weill Cornell Medical College of CornellUniversity, New York, NY, USA; Brain and Mind Research Institute, Weill CornellMedical College of Cornell University, New York, NY, USA

RUPASHREE DASS • Department of Chemistry, and Interdisciplinary Nanoscience Center(iNANO), Aarhus University, Aarhus, Denmark

GARY W. DAUGHDRILL • Department of Cell Biology, Microbiology, and Molecular Biology,University of South Florida, Tampa, FL, USA

FRANCESCA DIELLA • Structural and Computational Biology Unit, European MolecularBiology Laboratory, Heidelberg, Germany

HOLGER DINKEL • Max Planck Institute for Biological Cybernetics, Tubingen, GermanyA. KEITH DUNKER • Department of Biochemistry and Molecular Biology, Center for

Computational Biology and Bioinformatics, Indiana University School of Medicine,Indianapolis, IN, USA

RICHARD J. EDWARDS • School of Biotechnology and Biomolecular Sciences, University of NewSouth Wales, Sydney, NSW, Australia

DAVID ELIEZER • Department of Biochemistry, Weill Cornell Medical College of CornellUniversity, New York, NY, USA; Brain and Mind Research Institute, Weill CornellMedical College of Cornell University, New York, NY, USA

ALBERT ESCOBEDO • Institute for Research in Biomedicine (IRB Barcelona), The BarcelonaInstitute of Science and Technology, Barcelona, Spain; Joint BSC-IRBResearch Programmein Computational Biology, Barcelona, Spain

XIAO FAN • Department of Pediatrics, Columbia University, New York, NY, USAERNESTO FREIRE • Department of Biology, The Johns Hopkins University, Baltimore, MD,

USA; Department of Biology/Department of Biophysics and Biophysical Chemistry, ZanvylKrieger School of Arts & Sciences, Johns Hopkins University, Baltimore, MD, USA

JUAN A. GEREZ • Laboratory of Physical Chemistry, Department of Chemistry and AppliedBiosciences, ETH Zurich, Zurich, Switzerland

xiv Contributors

RANIA GHOUIL • Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS,Universite Paris-Saclay, Gif-sur-Yvette, France

STEFANO GIANNI • Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR,Sapienza Universita di Roma, Rome, Italy

TOBY J. GIBSON • Structural and Computational Biology Unit, European Molecular BiologyLaboratory, Heidelberg, Germany

GARRETT M. GINELL • Graduate Program in Biochemistry, Biophysics, and StructuralBiology, Division of Biological and Biomedical Sciences, Washington University in St. Louis,St. Louis, MO, USA; Department of Biomedical Engineering, Center for the Science andEngineering of Living Systems, Washington University in St. Louis, St. Louis, MO, USA

MARC GOUW • Structural and Computational Biology Unit, European Molecular BiologyLaboratory, Heidelberg, Germany

STEFFEN P. GRAETHER • Department of Molecular and Cellular Biology, University ofGuelph, Guelph, ON, Canada

ANDRAS HATOS • Department of Biomedical Sciences, University of Padua, Padua, ItalyRYO HAYAMA • Laboratory of Cellular and Structural Biology, The Rockefeller University,

New York, NY, USA; Panasonic Co., Secaucus, NJ, USAALEX S. HOLEHOUSE • Department of Biomedical Engineering, Center for the Science and

Engineering of Living Systems, Washington University in St. Louis, St. Louis, MO, USA;Department of Biochemistry and Molecular Biophysics, Washington University School ofMedicine, St. Louis, MO, USA

JACK HOLLAND • Department of Biochemistry, University of Oxford, Oxford, UKLINNEA ISAKSSON • Department of Chemistry and Molecular Biology, University of

Gothenburg, Gothenburg, SwedenYLVA IVARSSON • Department of Chemistry, BMC, Uppsala, SwedenKRISTINE STEEN JENSEN • Department for Biophysical Chemistry, Center for Molecular

Protein Science, LTH, Lund University, Lund, SwedenMANON JULIEN • Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS,

Universite Paris-Saclay, Gif-sur-Yvette, FranceGYORGY MIKLOS KESERU • Medicinal Chemistry Research Group, Research Centre for

Natural Sciences, Hungarian Academy of Sciences, Budapest, HungaryMAGNUS KJAERGAARD • Department of Molecular Biology and Genetics, Aarhus University,

Aarhus, Denmark; The Danish Research Institute for Translational Neuroscience(DANDRITE), Aarhus, Denmark; Center for Proteins in Memory—PROMEMO, DanishNational Research Foundation, Aarhus, Denmark

MANJEET KUMAR • Structural and Computational Biology Unit, European MolecularBiology Laboratory, Heidelberg, Germany

LUKASZ KURGAN • Department of Computer Science, Virginia Commonwealth University,Richmond, VA, USA

SARAH LEEB • Department of Biochemistry and Biophysics, Arrhenius Laboratories ofNatural Sciences, Stockholm University, Stockholm, Sweden

JULIE M. LETH • Finsen Laboratory, Rigshospitalet, The Biotech Research and InnovationCentre (BRIC), University of Copenhagen, Copenhagen, Denmark

KRESTEN LINDORFF-LARSEN • Department of Biology, Structural Biology and NMRLaboratory, Linderstrøm-Lang Centre for Protein Science, University of Copenhagen,Copenhagen N, Denmark

SARA LINSE • Biochemistry and Structural Biology, Lund University, Lund, Sweden

Contributors xv

MART LOOG • Institute of Technology, University of Tartu, Tartu, EstoniaALESSANDRA LUCHINI • Structural Biophysics, X-ray and Neutron Science, Niels Bohr

Institute, University of Copenhagen, Copenhagen, DenmarkFRANCESCA MALAGRINO • Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Istituto

Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari delCNR, Sapienza Universita di Roma, Rome, Italy

ARTEMI MALJAVIN • Institute of Technology, University of Tartu, Tartu, EstoniaERIK W. MARTIN • Department of Structural Biology, St. Jude Children’s Research Hospital,

Memphis, TN, USAMATTEO MASETTI • Department of Pharmacy and Biotechnology, Alma Mater Studiorum—

Universita di Bologna, Bologna, ItalyIRENA MATECKO-BURMANN • Department of Psychiatry and Neurochemistry, University of

Gothenburg, Goteborg, Sweden; Wallenberg Centre for Molecular and TranslationalMedicine, University of Gothenburg, Goteborg, Sweden

ATTILA MESZAROS • VIB Center for Structural Biology (CSB), Brussels, Belgium; StructuralBiology Brussels (SBB), Vrije Universiteit Brussel (VUB), Brussels, Belgium

SUSHAMA MICHAEL • Structural and Computational Biology Unit, European MolecularBiology Laboratory, Heidelberg, Germany

NICOLE M. MILKOVIC • Department of Structural Biology, St. Jude Children’s ResearchHospital, Memphis, TN, USA

TANJA MITTAG • Department of Structural Biology, St. Jude Children’s Research Hospital,Memphis, TN, USA

ANTHONY MITTERMAIER • Department of Chemistry, McGill University, Montreal, QC,Canada

ALEXANDER MIGUEL MONZON • Department of Biomedical Sciences, University of Padua,Padua, Italy

FRANS A. A. MULDER • Interdisciplinary Nanoscience Center (iNANO), Aarhus University,Aarhus C, Denmark; Department of Chemistry, Aarhus University, Aarhus C, Denmark

MARCO NECCI • Department of Biomedical Sciences, University of Padua, Padua, ItalyHUNG HUY NGUYEN • VIB Center for Structural Biology (CSB), Brussels, Belgium;

Structural Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), Brussels, BelgiumJAKOB T. NIELSEN • Interdisciplinary Nanoscience Center (iNANO), Aarhus University,

Aarhus C, Denmark; Department of Chemistry, Aarhus University, Aarhus C, DenmarkTIMOTHY J. NOTT • Department of Biochemistry, University of Oxford, Oxford, UKJANELLE NUNEZ-CASTILLA • Department of Biological Sciences, Biomolecular Sciences

Institute, Florida International University, Miami, FL, USACHRISTOPHER J. OLDFIELD • Department of Computer Science, Virginia Commonwealth

University, Richmond, VA, USAMIHKEL ORD • Institute of Technology, University of Tartu, Tartu, EstoniaKIRSTI PAULSEN • School of Biotechnology and Biomolecular Sciences, University of New South

Wales, Sydney, NSW, AustraliaKRIS PAUWELS • VIB Center for Structural Biology (CSB), Brussels, Belgium; Structural

Biology Brussels (SBB), Vrije Universiteit Brussel (VUB), Brussels, BelgiumBRANDON PAYLISS • Department of Biochemistry, University of Toronto, Toronto, ON,

CanadaANDERS PEDERSEN • Swedish NMR Centre, University of Gothenburg, Gothenburg, Sweden

xvi Contributors

CHRISTIAN PARSBÆK PEDERSEN • Department of Biology, Structural Biology and NMRLaboratory and the Linderstrøm-Lang Centre for Protein Science, University ofCopenhagen, Copenhagen N, Denmark

IVAN PERAN • Department of Structural Biology, St. Jude Children’s Research Hospital,Memphis, TN, USA

ASA PEREZ-BERCOFF • School of Biotechnology and Biomolecular Sciences, University of NewSouth Wales, Sydney, NSW, Australia

LASZLO PETRI • Medicinal Chemistry Research Group, Research Centre for Natural Sciences,Hungarian Academy of Sciences, Budapest, Hungary

DAMIANO PIOVESAN • Department of Biomedical Sciences, University of Padua, Padua, ItalyMICHAEL PLOUG • Finsen Laboratory, Rigshospitalet, The Biotech Research and Innovation

Centre (BRIC), University of Copenhagen, Copenhagen, DenmarkNATALIA C. PRYMACZOK • Laboratory of Physical Chemistry, Department of Chemistry and

Applied Biosciences, ETH Zurich, Zurich, SwitzerlandDAVID F. RENDON-LUNA • Departamento de Biologıa Molecular de Plantas, Instituto de

Biotecnologıa, Universidad Nacional Aut�onoma de Mexico, Cuernavaca, Morelos, MexicoJOSE L. REYES • Departamento de Biologıa Molecular de Plantas, Instituto de Biotecnologıa,

Universidad Nacional Aut�onoma de Mexico, Cuernavaca, Morelos, MexicoELIZABETH RHOADES • Graduate Group in Biochemistry and Molecular Biophysics, Perelman

School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department ofChemistry, University of Pennsylvania, Philadelphia, PA, USA

ROLAND RIEK • Laboratory of Physical Chemistry, Department of Chemistry and AppliedBiosciences, ETH Zurich, Zurich, Switzerland

ELLEN RIELOFF • Division of Theoretical Chemistry, Department of Chemistry, LundUniversity, Lund, Sweden

PAULETTE S. ROMERO-PEREZ • Departamento de Biologıa Molecular de Plantas, Instituto deBiotecnologıa, Universidad Nacional Aut�onoma de Mexico, Cuernavaca, Morelos, Mexico

MICHAEL P. ROUT • Laboratory of Cellular and Structural Biology, The RockefellerUniversity, New York, NY, USA

KIERSTEN M. RUFF • Department of Biomedical Engineering, Washington University in St.Louis, St. Louis, MO, USA

AMIN SAGAR • Centre de Biochimie Structurale, INSERM, CNRS, Universite de Montpellier,Montpellier, France

XAVIER SALVATELLA • Institute for Research in Biomedicine (IRB Barcelona), The BarcelonaInstitute of Science and Technology, Barcelona, Spain; Joint BSC-IRBResearch Programmein Computational Biology, Barcelona, Spain; ICREA, Barcelona, Spain

ARNE SCHON • Department of Biology, The Johns Hopkins University, Baltimore, MD, USAPERNILLE SEIFFERT • Department of Biology, Structural Biology and NMR Laboratory and

the Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, CopenhagenN, Denmark

JESSICA SILTBERG-LIBERLES • Department of Biological Sciences, Biomolecular SciencesInstitute, Florida International University, Miami, FL, USA

LEANDRO SIMONETTI • Department of Chemistry, BMC, Uppsala, SwedenKARAMJEET K. SINGH • Department of Molecular and Cellular Biology, University of Guelph,

Guelph, ON, CanadaMARIE SKEPO • Division of Theoretical Chemistry, Department of Chemistry, Lund

University, Lund, Sweden

Contributors xvii

CHARLOTTE S. SØRENSEN • Department of Molecular Biology and Genetics, AarhusUniversity, Aarhus, Denmark; The Danish Research Institute for TranslationalNeuroscience (DANDRITE), Aarhus, Denmark

SAMUEL SPARKS • Department of Biochemistry, Albert Einstein College of Medicine, Bronx,NY, USA; Silicon Therapeutics, Boston, MA, USA

DMITRI SVERGUN • European Molecular Biology Laboratory, Hamburg Unit, EMBL c/oDESY, Hamburg, Germany

FRANCOIS-XAVIER THEILLET • Institute for Integrative Biology of the Cell (I2BC), CEA,CNRS, Universite Paris-Saclay, Gif-sur-Yvette, France

PETER TOMPA • VIB Center for Structural Biology (CSB), Brussels, Belgium; StructuralBiology Brussels (SBB), Vrije Universiteit Brussel (VUB), Brussels, Belgium; Institute ofEnzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences,Budapest, Hungary

SILVIO C. E. TOSATTO • Department of Biomedical Sciences, University of Padua, Padua,Italy; CNR Institute of Neuroscience, Padua, Italy

ANGELO TOTO • Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Istituto Pasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR,Sapienza Universita di Roma, Rome, Italy

FRANCESCA TROILO • Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, IstitutoPasteur-Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari delCNR, Sapienza Universita di Roma, Rome, Italy

VLADIMIR N. UVERSKY • Department of Molecular Medicine, Morsani College of Medicine,University of South Florida, Tampa, FL, USA; USF Health Byrd Alzheimer’s ResearchInstitute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA;Laboratory of New Methods in Biology, Institute for Biological Instrumentation, RussianAcademy of Sciences, Pushchino, Moscow Region, Russian Federation

ERVIN VALK • Institute of Technology, University of Tartu, Tartu, EstoniaGUY VANDENBUSSCHE • Laboratory for the Structure and Function of Biological Membranes,

Centre for Structural Biology and Bioinformatics, Universite Libre de Bruxelles (ULB),Brussels, Belgium

KIM VAN ROEY • Structural and Computational Biology Unit, European Molecular BiologyLaboratory, Heidelberg, Germany

CHEN WANG • Department of Medicine, Columbia University, New York, NY, USACHRISTOPHER A. WAUDBY • Institute of Structural and Molecular Biology, UCL and

Birkbeck College, London, UKSANJULA P. WICKRAMASINGHE • Graduate Group in Biochemistry and Molecular Biophysics,

Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USAPETER E. WRIGHT • Department of Integrative Structural and Computational Biology, The

Scripps Research Institute, La Jolla, CA, USAKE YANG • Department of Integrative Structural and Computational Biology, The Scripps

Research Institute, La Jolla, CA, USASOPHIE ZINN-JUSTIN • Universite Paris-Saclay, CEA, CNRS, Institute for Integrative

Biology of the Cell (I2BC), Gif-sur-Yvette, France

xviii Contributors