Isoprenoid-chained lipid EROCOC 17+4 : a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography

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...

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Bibliographic Details
Published in:Scientific reports 2020-11, Vol.10 (1), p.19305
Main Authors: Ihara, Kentaro, Hato, Masakatsu, Nakane, Takanori, Yamashita, Keitaro, Kimura-Someya, Tomomi, Hosaka, Toshiaki, Ishizuka-Katsura, Yoshiko, Tanaka, Rie, Tanaka, Tomoyuki, Sugahara, Michihiro, Hirata, Kunio, Yamamoto, Masaki, Nureki, Osamu, Tono, Kensuke, Nango, Eriko, Iwata, So, Shirouzu, Mikako
Format: Article
Language:English
Subjects:
Animals
Cholesterol - chemistry
Crystallization
Crystallography, X-Ray - instrumentation
Escherichia coli
Lipids - chemistry
Membrane Proteins - chemistry
Monoglycerides - chemistry
Receptors, Adenosine A2 - chemistry
Sf9 Cells
Spodoptera
Synchrotrons
Temperature
Terpenes - chemistry
X-Rays
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Summary: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 EROCOC 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 A receptor (A R). The structures of EROCOC -matrix grown A R 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 A R crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC /water system did not form the crystalline L phase at least down to - 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline L phase below about 17 °C. As the L phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC will expand the utility of the in meso method.
ISSN:2045-2322