Harnessing the power of an X-ray laser for serial crystallography of membrane proteins crystallized in lipidic cubic phase.

Ming-Yue Lee,Sébastien Boutet,Anton Barty,Uwe Weierstall,Cornelius Gati,Thomas A White,Vadim Cherezov,Wei Liu,Andrii Ishchenko,Andrew Aquila,James Geiger,Mark S Hunter,Gye Won Han,Alexander Batyuk

doi:10.1107/s2052252520012701

Abstract

Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; however, like most techniques, it has limitations. Here we attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method. Using an optimized SFX experimental setup in a helium atmosphere, the room-temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution is determined and compared with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, the capability of utilizing high XFEL beam transmissions is demonstrated, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete dataset. The experimental setup presented herein can be applied to future SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.

Highlights

Elucidating high-resolution X-ray structures of G-proteincoupled receptors (GPCRs) and other membrane proteins using synchrotron radiation sources has been limited by the difficulty of obtaining high-quality crystals that can withstand radiation damage
Serial femtosecond crystallography (SFX) with X-ray free-electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase; like most techniques, it has limitations
We attempt to address some of these limitations related to the use of a vacuum chamber and the need for attenuation of the XFEL beam, in order to further improve the efficiency of this method

Summary

Introduction

Elucidating high-resolution X-ray structures of G-proteincoupled receptors (GPCRs) and other membrane proteins using synchrotron radiation sources has been limited by the difficulty of obtaining high-quality crystals that can withstand radiation damage. Several challenges must be overcome during crystallization and diffraction data collection to achieve high-resolution structure models. The size of a protein crystal suitable to resolve a 3.5 Astructural model using synchrotron diffraction should be at least 20 mm in each dimension (Sliz et al, 2003). As secondary radiation damage propagates throughout the crystals, diffraction data quality deteriorates, resulting in decreased resolution, and increased unit-cell volume, B factors and mosaicity (Garman & Owen, 2006).

Methods

Results

Conclusion