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

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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.
Funding Information
  • National Institute of General Medical Sciences (R35 GM127086, R01 GM124152)
  • National Institute on Drug Abuse (R21 DA042298)
  • BioXFEL Science and Technology Center (1231306)
  • National Science Foundation
  • Deutsches Elektronen-Synchrotron
  • SLAC National Accelerator Laboratory

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