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

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 A_2A receptor (A_2AAR) at 2.0 Å resolution is determined and compared with previous A_2AAR 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.

Authors:

Lee, Ming-Yue ;

• Search DOE PAGES for author "Lee, Ming-Yue"
• Search DOE PAGES for ORCID "0000-0001-7082-1216"

Geiger, James; Ishchenko, Andrii ;

• Search DOE PAGES for author "Ishchenko, Andrii"
• Search DOE PAGES for ORCID "0000-0002-3797-0604"

Han, Gye Won ;

• Search DOE PAGES for author "Han, Gye Won"
• Search DOE PAGES for ORCID "0000-0003-4107-835X"

Barty, Anton; White, Thomas A.; Gati, Cornelius ;

• Search DOE PAGES for author "Gati, Cornelius"
• Search DOE PAGES for ORCID "0000-0003-3693-7009"

Batyuk, Alexander; Hunter, Mark S.; Aquila, Andrew; Boutet, Sébastien ;

• Search DOE PAGES for author "Boutet, Sébastien"
• Search DOE PAGES for ORCID "0000-0003-3928-1244"

Weierstall, Uwe; Cherezov, Vadim ;

• Search DOE PAGES for author "Cherezov, Vadim"
• Search DOE PAGES for ORCID "0000-0002-5265-3914"

Liu, Wei

Publication Date:

Thu Oct 15 00:00:00 EDT 2020

Research Org.:

SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); National Science Foundation (NSF)

OSTI Identifier:

1675009

Alternate Identifier(s):

OSTI ID: 1768894

Grant/Contract Number:  

AC02-76SF00515; R35 GM127086; R01 GM124152; R21 DA042298; 1231306

Resource Type:

Published Article

Journal Name:

IUCrJ

Additional Journal Information:

Journal Name: IUCrJ Journal Volume: 7 Journal Issue: 6; Journal ID: ISSN 2052-2525

Publisher:

International Union of Crystallography

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; G-protein-coupled receptors; membrane proteins; XFELs; serial femtosecond crystallography; adenosine A2A receptors; lipidic cubic phases; high dynamic range detectors.