Serial Femtosecond Crystallography of G Protein-Coupled Receptors

Serial Femtosecond Crystallography of G Protein-CoupledReceptors

Wei Liu1, Daniel Wacker1, Cornelius Gati2, Gye Won Han1, Daniel James3, Dingjie Wang3,Garrett Nelson3, Uwe Weierstall3, Vsevolod Katritch1, Anton Barty2, Nadia A. Zatsepin3,Dianfan Li4, Marc Messerschmidt5, Sébastien Boutet5, Garth J. Williams5, Jason E.Koglin5, M. Marvin Seibert5,6, Chong Wang1, Syed T.A. Shah4, Shibom Basu7, RaimundFromme7, Christopher Kupitz7, Kimberley N. Rendek7, Ingo Grotjohann7, Petra Fromme7,Richard A. Kirian2,3, Kenneth R. Beyerlein2, Thomas A. White2, Henry N. Chapman2,8,9,Martin Caffrey4, John C.H. Spence3, Raymond C. Stevens1, and Vadim Cherezov1,*

1Department of Integrative Structural and Computational Biology, The Scripps Research Institute,La Jolla, CA 92037, USA. 2Center for Free Electron Laser Science, DESY, 22607 Hamburg,Germany. 3Department of Physics, Arizona State University, Tempe, AZ 85287, USA. 4School ofMedicine and School of Biochemistry and Immunology, Trinity College, Dublin, Dublin 2, Ireland.5SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.6Laboratory of Molecular Biophysics, Department of Cell and Molecular Biology, UppsalaUniversity, Husargatan 3 (Box 596), SE-751 24 Uppsala, Sweden. 7Department of Chemistry andBiochemistry, Arizona State University, Tempe, AZ 85287, USA. 8Institute for ExperimentalPhysics, University of Hamburg, 22761 Hamburg, Germany. 9Center for Ultrafast Imaging, 22607Hamburg, Germany.

*Correspondence to:[email protected].

Author contributions:W.L. developed protocols of producing high density microcrystals in LCP, prepared samples, helped with testing LCP injector, datacollection and writing the paper.Da.W. prepared 5-HT2B microcrystals in LCP, helped with data collection, structure refinement, analysis and writing the paper.C.G. participated in data collection, processed and analyzed data.G.W.H. performed structure refinement.D.J., Di.W. and G.N. helped develop and operate the LCP injector.U.W. conceived, designed and developed the LCP injector.V.K. analyzed the results and helped with writing the paper.A.B. participated in data collection, wrote data processing software and helped with data processing and with writing the paper.N.A.Z., Sh.B. participated in data collection, helped with data processingD.L. helped with data collection.Se.B., M.M., G.J.W., J.E.K., M.M.S. setup the x-ray FEL experiment, beamline, controls and data acquisition, operated the CXIbeamline and performed the data collection.C.W. helped with sample preparation.S.T.A.S. synthesized and purified 7.9 MAG.R.F., C.K., K.N.R., I.G. participated in data collection and contributed to sample characterization.P.F. was involved in the initiation and planning of the experiments, assisted with sample characterization, data collection andcontributed to writing the paper.R.A.K. developed the Monte Carlo integration method, contributed to data processing.K.R.B. contributed to software development and data processing.T.A.W. developed the Monte Carlo integration method, wrote data processing software, contributed to data processing.H.N.C. supervised software development and data processing, helped with writing the paper.M.C. provided the 7.9 MAG, helped with data collection and with writing the paper.J.C.H.S. helped develop the LCP injector and developed the Monte Carlo integration method with R.A.K.R.C.S. supervised GPCR production and contributed to writing the paper.V.C. conceived the project, designed the experiments, supervised data collection, performed structure refinement, analyzed the resultsand wrote the paper.

NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2014 June 20.

Published in final edited form as:Science. 2013 December 20; 342(6165): 1521–1524. doi:10.1126/science.1244142.

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AbstractX-ray crystallography of G protein-coupled receptors and other membrane proteins is hamperedby difficulties associated with growing sufficiently large crystals that withstand radiation damageand yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser(XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained ahigh-resolution room temperature structure of a human serotonin receptor using sub-10 µmmicrocrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared tothe structure solved by traditional microcrystallography from cryo-cooled crystals of about twoorders of magnitude larger volume, the room temperature XFEL structure displays a distinctdistribution of thermal motions and conformations of residues that likely more accuratelyrepresent the receptor structure and dynamics in a cellular environment.

G protein-coupled receptors (GPCRs) represent a highly diverse superfamily of eukaryoticmembrane proteins that mediate cellular communication. In humans, about 800 GPCRsrespond to a variety of extracellular signaling molecules and transmit signals inside the cellby coupling to heterotrimeric G proteins and other effectors. Their involvement in keyphysiological and sensory processes in humans makes GPCRs prominent drug targets.Despite the high biomedical relevance and decades of dedicated research, knowledge of thestructural mechanisms of ligand recognition, receptor activation and signaling in this broadfamily remains limited. Challenges for GPCR structural studies include low expressionyields, low receptor stability after detergent extraction from native membranes, and highconformational heterogeneity. Many years of developments aimed at receptor stabilization,crystallization and microcrystallography culminated in a series of breakthroughs in GPCRstructural biology leading to the structure determination of 22 receptors, some of which weresolved in several conformational states and one in complex with its G protein partner (1–5).

Nonetheless, crystallographic studies of GPCRs remain difficult, as many of them produceonly microcrystals. Most GPCR structures to date have been obtained using crystallizationfrom the membrane-mimetic environment of a lipidic cubic phase (LCP) (6, 7). LCPcrystallization has proven successful for obtaining high-resolution structures of a variety ofmembrane proteins including ion channels, transporters, and enzymes, in addition to GPCRs(8, 9). This method leads to highly ordered crystals that are, however, often limited in size.Microfocus x-ray beams of high intensity (~109 photons/s/µm2) and long exposures (~5 s)are typically required to obtain sufficient intensity for high-resolution data from weaklydiffracting microcrystals. The high radiation doses induce severe radiation damage andrequire merging data from multiple crystals to obtain complete datasets of sufficient quality.Accordingly, sub-10 µm GPCR crystals are currently not suitable for high-resolution datacollection even at the most powerful synchrotron microfocus beamlines (7, 10).

Serial femtosecond crystallography (SFX) (11), which takes advantage of x-ray free-electronlasers (XFEL), has recently demonstrated great promise for obtaining room temperaturehigh-resolution data from micrometer- and sub-micrometer size crystals of soluble proteinswith minimal radiation damage (12, 13). The highly intense (~2 mJ, 1012 photons per pulse)and ultrashort (<50 fs) x-ray pulses produced by XFELs enable recording high-resolutiondiffraction snapshots from individual crystals at single orientations before their destruction.SFX data collection, therefore, relies on a continuous supply of small crystals intersectingthe XFEL beam in random orientations, typically provided by a fast-running liquid microjet(12), which is incompatible with streaming highly viscous gel-like materials, such as LCP,and requires tens to hundreds milligrams of crystallized protein for data collection (11). Formany membrane proteins, including most human membrane proteins, obtaining suchquantities is not practical.

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Here we have modified the SFX data collection approach (Fig. 1) and obtained a room-temperature GPCR structure at 2.8 Å resolution using only 300 µg of protein crystallized inLCP. SFX experiments were performed at the Coherent X-ray Imaging (CXI) instrument ofthe Linac Coherent Light Source (LCLS) (14). LCP-grown microcrystals (average size5×5×5 µm3) (fig. S1) (15) of the human serotonin 5-HT2B receptor (16) bound to the agonistergotamine were continuously delivered across a ~1.5 µm diameter XFEL beam, using aspecially designed LCP injector. LCP with randomly distributed crystals was extrudedthrough a 20–50 µm capillary into a vacuum chamber (10−4 Torr) at room temperature (21°C) (17) and a constant flow-rate of 50 – 200 nL/min, and was stabilized by a co-axial flowof helium or nitrogen gas supplied at 300–500 psi. Single-pulse diffraction patterns (fig. S2)were recorded using 9.5 keV (1.3 Å) x-ray pulses of 50 fs duration at a 120 Hz repetitionrate by a Cornell-SLAC pixel array detector (CSPAD) (18) positioned at a distance of 100mm from the sample. The XFEL beam was attenuated to 3–6% to avoid detector saturation.The average x-ray pulse energy at the sample was 50 µJ (3·1010 photons/pulse),corresponding to a radiation dose of up to 25 MGy per crystal. A total of 4,217,508diffraction patterns were collected within 10 h using ~100 µL of crystal-loaded LCP,corresponding to about 0.3 mg of protein. Of these patterns, 152,651 were identified ascrystal hits (15 or more Bragg peaks) by the processing software Cheetah (19),corresponding to a hit rate of 3.6 %. Of these crystal hits, 32,819 patterns (21.5 %) weresuccessfully indexed and integrated by CrystFEL (20) at 2.8 Å resolution (table S1). Thestructure was determined by molecular replacement and refined to Rwork/Rfree = 22.7%/27.0%. Overall, the final structure (fig. S3) has a well-defined density for most residuesincluding the ligand ergotamine (fig. S4).

The XFEL structure of the 5-HT2B receptor/ergotamine complex (5-HT2B-XFEL) wascompared to the recently published structure of the same receptor/ligand complex obtainedby traditional microcrystallography at a synchrotron source (PDB ID: 4IB4; 5-HT2B-SYN)(21). Synchrotron data were collected at 100 K on cryo-cooled crystals of a much larger size(average volume ~104 µm3) than those used for the XFEL structure (average volume ~102

µm3) (fig. S1). Other differences between data collection protocols are listed in table S1.Both datasets were processed in the same spacegroup C2221, which is expected given thevery similar crystallization conditions. However, the lattice parameters for the roomtemperature XFEL crystals are slightly longer in the a and b directions and slightly shorterin the c direction, resulting in a 2.1% larger unit cell volume. Concomitant with these latticechanges we observe a ~2.5° rotation of the BRIL fusion domain with respect to the receptor(fig. S5). Otherwise, the receptor domains of the 5-HT2B-XFEL and 5-HT2B-SYN structuresare very similar (receptor Cα root mean square deviation (rmsd) = 0.45 Å, excludingflexible residues at the N-terminus, 48–51, and in the extracellular loop (ECL) 2, 195–205)(Fig. 2). The ligand ergotamine has indistinguishable electron density and placement (totalligand rmsd = 0.32 Å) in both structures (Fig. 2, fig. S4B). The largest backbone deviationsare observed in the loop regions, especially in the stretch of ECL2 between helix IV and theCys128 – Cys207 disulfide bond, which is apparently very flexible. An unexpectedbackbone deviation is observed at the extracellular tip of helix II (Fig. 2C), which adopts aregular α-helix in the 5-HT2B-XFEL structure with Thr114 forming a stabilizing hydrogenbond with the main chain hydroxyl of Ile110. In the 5-HT2B-SYN structure, however, a waterstabilized kink is found at this location, which results in the two structures deviating by 2.0Å (at Cα atom of Thr114) at the tip of helix II and up to 3.4 Å (at O atom of Phe117) inECL1.

Although absolute B- (or temperature) factor values can be affected by errors associatedwith experimental conditions, their distribution generally represents the relative static anddynamic flexibility of the protein in the crystal (22). Since both structures were obtainedfrom similar samples and at similar resolutions, we analyzed their B-factor distributions to

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study the effect of the different temperatures on the thermal motions of the receptor. Theaverage B-factor for the receptor part in the room temperature 5-HT2B-XFEL structure (88.4Å2) is 21 Å2 larger than that in the cryo 5-HT2B-SYN structure (67.2 Å2), consistent withlarger thermal motions at higher temperature and possible effects of Bragg terminationduring the XFEL pulse (20). The distribution of B-factors highlights a more rigid core of theseven transmembrane helices in comparison to loops, with more pronounced B-factordeviations observed in the room temperature 5-HT2B-XFEL structure (Fig. 3, fig. S6). N-terminus, intracellular loop (ICL) 2, ECL1, and part of ECL2 between helix IV and theCys128 – Cys207 disulfide bond show much larger deviations in B-factors (50–100 Å2)between the two structures compared to the average difference of 21 Å2. These parts of thestructure are not involved in direct interactions with the ligand ergotamine, but theirmobility may affect the kinetics of ligand binding and interactions with intracellular bindingpartners (23). In contrast, ICL1, part of ECL2 between the Cys128 – Cys207 disulfide bondand helix V, and ECL3 display just an average increase in the B-factors, suggesting that therelative range of their thermal fluctuations was adequately captured in the cryo structure. Aspreviously established by cryocrystallography, one of the most pronounced differencesbetween the two subtypes of serotonin receptors, 5-HT2B and 5-HT1B, occurs at theextracellular tip of helix V and ECL2, which forms an additional helical turn stabilized by awater molecule in 5-HT2B (21). This additional turn pulls the extracellular tip of helix Vtoward the center of the helical bundle, and was suggested to be responsible for the biasedagonism of ergotamine at the 5-HT2B receptor. The 5-HT2B-XFEL structure confirms therigid structured conformation of ECL2, stabilized by a comprehensive network of hydrogenbonds, involving residues Lys193, Glu196, Arg213, Asp216 and a lipid OLC (monoolein)(fig. S7), however, no ordered water molecule is observed, emphasizing that water is moredisordered and probably does not play a substantial structural role at this location.

Several side chains have partly missing electron density in both room temperature and cryostructures (table S2). Such lack of density is most likely related to disorder of thecorresponding side chains (such as residues at the N-terminus, ECL2 and ICL2) (Fig. 2A).Two disulfide bonds, Cys128 – Cys207 and Cys350 – Cys353, are intact and well resolvedin both structures, however the B-factor increase in the 5-HT2B-XFEL structure compared to5-HT2B-SYN for each of these disulfide bonds (11.1 Å2 and 5.7 Å2, respectively) is lowerthan the average B-factor increase (21 Å2). Several side chains have different rotamerconformations between the two structures (table S3, Fig. 2D), consistent with a partialremodeling of the side chain conformational distribution upon cryo-cooling observed insoluble proteins (24). Interestingly, several interactions involving charged residues appearstronger and better defined in 5-HT2B-XFEL compared to the 5-HT2B-SYN structure (tableS4). This strengthening of the charged interactions at higher temperatures potentially can beexplained by a decrease in the dielectric constant of water with temperature, reducing the de-solvation penalty (25, 26). In particularly, the salt bridge between Glu319 and Lys247 iswell defined in the 5-HT2B-XFEL structure, but appears broken in the cryo 5-HT2B-SYNstructure (Fig. 2B). Since GPCR activation has been associated with large-scale structuralchanges in the intracellular parts of helices V and VI, this salt bridge may play a role in thereceptor function and is likely to be more accurately resolved and represented in the 5-HT2B-XFEL structure recorded at room temperature.

Overall, the observed differences likely originate from effects related to thermal motions,cryo-cooling (24) and radiation damage (27). Thus, the XFEL source enables access to aroom temperature GPCR structure, which more accurately represents the conformationalensemble for this receptor under native conditions. Since dynamics are an integral part ofGPCR biology, the use of SFX to accurately determine GPCR structural details at roomtemperature can make an important contribution to understanding the structure-functionrelationships in this superfamily.

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Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsParts of this research were carried out at the LCLS, a National User Facility operated by Stanford University onbehalf of the U.S. Department of Energy, Office of Basic Energy Sciences and at the GM/CA CAT of the ArgonnePhoton Source, Argonne National Laboratory. This work was supported by the National Institutes of HealthCommon Fund in Structural Biology grants P50 GM073197 (V.C. and R.C.S.), P50 GM073210 (M.C.), R01GM095583 (P.F.); NIGMS PSI:Biology grants U54 GM094618 (V.C., V.K. and R.C.S.), U54 GM094599 (P.F.)and NSF STC award 1231306 (J.C.H.S.). We further acknowledge support from the Helmholz Association, theGerman Research Foundation (DFG), and the German Federal Ministry of Education and Research (BMBF)(H.N.C.), and from Science Foundation Ireland (07/IN.1/B1836, 12/IA/1255) (M.C.).

Special thanks to G. M. Stewart, T. Anderson, and SLAC Infomedia and K. Kadyshevskaya from TSRI forpreparing Fig. 1, T. Trinh and M. Chu for help with baculovirus expression, H. Liu and M. Klinker for help withdata processing, A. Walker for assistance with manuscript preparation, and I. Wilson for reviewing the manuscript.

Coordinates and the structure factors have been deposited in the Protein Data Bank under the accession code 4NC3.The diffraction patterns have been deposited in the Coherent X-ray Imaging Data Bank cxidb.org under theaccession code ID-21.

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16. The engineered for crystallization construct is based on the sequence of the human 5-HT2Breceptor with the following modifications: (a) Residues Tyr249-Val313 within ICL3 were replacedwith Ala1-Leu106 of thermostabilized apo cytochrome b562 RIL (BRIL), (b) N-terminal residues1–35 and C-terminal residues 406–481 were truncated, and (c) a thermostabilizing M144Wmutation was introduced.

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Fig. 1. Experimental setup for SFX data collection using an LCP injector5-HT2B receptor microcrystals (first zoom level) dispersed in LCP (second zoom level) areinjected as a continuous column of 20–50 µm diameter inside a vacuum chamber andintersected with 1.5 µm diameter pulsed XFEL beam focused by Kirkpatrick-Baez (K-B)mirrors. Single pulse diffraction patterns are collected at 120 Hz using a CSPAD detector.

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Fig. 2. Comparison between 5-HT2B-XFEL (light red) and 5-HT2B-SYN (cyan) structuresCentral image represents a backbone overlay of the two structures. Dashed lines correspondto membrane boundaries defined by the Orientation of Proteins in Membrane database(http://opm.pharm.umich.edu) (28). (A) Electron density for the Glu212 side chain ismissing in 5-HT2B-SYN and fully resolved in 5-HT2B-XFEL. (B) A salt bridge betweenGlu319 and Lys247 links intracellular parts of helices V and VI in the 5-HT2B-XFELstructure. In the 5-HT2B-SYN structure Lys247 makes a hydrogen bond with Tyr1105 fromthe BRIL fusion protein. (C) Extracellular tip of helix II forms a regular helix in 5-HT2B-XFEL with Thr114 making a stabilizing hydrogen bond with the backbone carbonyl,while in 5-HT2B-SYN a water stabilized kink is introduced at this position. (D) Tyr87 forms ahydrogen bond with Asn90 in 5-HT2B-XFEL; this hydrogen bond is broken and Tyr87 adoptsa different rotamer conformation in 5-HT2B-SYN structure. 2mFobs-Fcalc maps (contoured at1 σ level) are shown only around described residues.

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http://opm.pharm.umich.edu/

Fig. 3. Differences in B-factors between 5-HT2B-XFEL and 5-HT2B-SYN structures(A) B-factors difference (BXFEL-BSYN) for Cα atoms plotted vs. residue number. (B) Viewof the 5-HT2B-XFEL structure from the extracellular side and (C) in the lateral to membraneorientation. Structure in B and C is shown in putty representation and colored in rainbowcolors by the Cα B-factors (range 60 – 170 Å2). Loops for which B-factors increased aboveaverage are labeled in red and those that have about an average increase are labeled in bluein (A) and (C). Helices are labeled in (B).

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Serial Femtosecond Crystallography of G Protein-Coupled Receptors