Native phasing of x-ray free-electron laser data for a G protein-coupled receptor

Alexander Batyuk¹, Lorenzo Galli², Andrii Ishchenko³, Gye Won Han³, Cornelius Gati², Petr A Popov⁴, Ming-Yue Lee³, Benjamin Stauch³, Thomas A White², Anton Barty², Andrew Aquila⁵, Mark S Hunter⁵, Mengning Liang⁵, Sébastien Boutet⁵, Mengchen Pu⁶, Zhi-Jie Liu⁷, Garrett Nelson⁸, Daniel James⁸, Chufeng Li⁸, Yun Zhao⁸, John C H Spence⁸, Wei Liu⁹, Petra Fromme⁹, Vsevolod Katritch¹⁰, Uwe Weierstall⁸, Raymond C Stevens¹¹, Vadim Cherezov¹²

Affiliations

¹ Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
² Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.
³ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.
⁴ Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
⁵ Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
⁶ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
⁷ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.; iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
⁸ Department of Physics, Arizona State University, Tempe, AZ 85287, USA.
⁹ Center for Applied Structural Discovery at the Biodesign Institute, School of Molecular Sciences, Arizona State University, 727 East Tyler Street, Tempe, AZ 85287, USA.
¹⁰ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
¹¹ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.; iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
¹² Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.; Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089, USA.

PMID: 27679816
PMCID: PMC5035125
DOI: 10.1126/sciadv.1600292

Native phasing of x-ray free-electron laser data for a G protein-coupled receptor

Alexander Batyuk et al. Sci Adv. 2016.

. 2016 Sep 23;2(9):e1600292.

doi: 10.1126/sciadv.1600292. eCollection 2016 Sep.

Authors

Affiliations

¹ Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
² Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607 Hamburg, Germany.
³ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.
⁴ Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
⁵ Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
⁶ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
⁷ National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.; iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
⁸ Department of Physics, Arizona State University, Tempe, AZ 85287, USA.
⁹ Center for Applied Structural Discovery at the Biodesign Institute, School of Molecular Sciences, Arizona State University, 727 East Tyler Street, Tempe, AZ 85287, USA.
¹⁰ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
¹¹ Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.; iHuman Institute, ShanghaiTech University, Shanghai 201210, China.
¹² Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.; Bridge Institute, University of Southern California, Los Angeles, CA 90089, USA.; Moscow Institute of Physics and Technology, Dolgoprudny 141700, Russia.; Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.; Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089, USA.

PMID: 27679816
PMCID: PMC5035125
DOI: 10.1126/sciadv.1600292

Abstract

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of "diffraction-before-destruction." However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A_2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

Keywords: Crystallography; GPCR; SAD; de novo structure; lipidic cubic phase; native phasing; protein; serial femtosecond crystallography; sulfur; x-ray free electron laser.

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Figures

**Fig. 1. Sulfur peaks in the anomalous difference A_2AAR Fourier map.**
Sulfur density is contoured at 3 σ and overlaid on the A_2AAR crystal structure. Twenty sulfur atoms could be identified from the map. BRIL fusion moiety containing one ordered sulfur atom (M1033) is not shown. Three sulfurs (M-24, C-13, and M1058) are disordered and do not have electron density.

**Fig. 2. Improvements in electron density at different stages of the phasing process.**
(A) Phaser EP map. (B) Resolve density modified map. (C) Autobuild autotraced map. Omit electron density around the ligand is shown on the top panels. 2mF_o-DF_c electron density map for helix III is shown on the bottom panels. All maps are contoured at 1.0 σ.

**Fig. 3. Comparison of resolved water molecules between the room temperature XFEL structure (A_2A_S-SAD_1.9) and the cryocooled synchrotron structure (PDB: 4EIY).**
(A) Cartoon representation of the XFEL structure with overlaid waters. Water molecules from the XFEL structure are shown as semitransparent spheres, whereas waters from PDB: 4EYI are shown as dots, colored by location: green, close proximity to ligand (<5 Å); red, sodium ion pocket (<10 Å); cyan, other regions. (B) Conservation of the water positions between PDB: 4EIY and XFEL structures. For each water molecule in PDB: 4EIY, the distance to the closest water in the XFEL structure is shown on the y axis, whereas its B factor is shown on the x axis. Data points are colored the same way as in (A). Positions of water molecules can be considered as conserved if the distance between corresponding water molecules in two structures is less than 1 Å.

See this image and copyright information in PMC

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Native phasing of x-ray free-electron laser data for a G protein-coupled receptor

Affiliations

Native phasing of x-ray free-electron laser data for a G protein-coupled receptor

Authors

Affiliations

Abstract

Figures

References

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources