Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography

Kentaro Ihara, Masakatsu Hato, Takanori Nakane, Keitaro Yamashita, Tomomi Kimura-Someya, Toshiaki Hosaka, Yoshiko Ishizuka-Katsura, Rie Tanaka, Tomoyuki Tanaka, Michihiro Sugahara, Kunio Hirata, Masaki Yamamoto, Osamu Nureki, Kensuke Tono, Eriko Nango, So Iwata, Mikako Shirouzu

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Abstract

In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

Original languageEnglish
Article number19305
JournalScientific reports
Volume10
Issue number1
DOIs
Publication statusPublished - 2020 Dec
Externally publishedYes

ASJC Scopus subject areas

  • General

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