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

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

doi:10.1038/s41598-020-76277-x

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.

Highlights

In meso crystallization of membrane proteins (MPs), a powerful technique for MP structure determination, critically relies on the choice of the lipids that form the lipidic cubic phase (LCP) around room temperature[1,2]
We demonstrate that EROCOC17+4-matrix, a 9:1 (w/w) mixture of EROCOC17+4 and cholesterol, supports the crystallization of A 2AR at 20 °C, yielding a high-resolution crystal structure comparable to that of the MO-matrix grown crystals at 1.8 Å resolution (PDB ID: 4EIY, hereafter referred to as the 4EIY model), and that EROCOC17+4-matrix allows us to perform the LCP-SFX data collection with a sample kept at 4 °C, without the LC phase formation
This study highlights the use of E ROCOC17+4 as a new matrix lipid for A2AR crystallization and an A2AR crystal delivery medium in LCP-SFX

Summary

Introduction

In meso crystallization of membrane proteins (MPs), a powerful technique for MP structure determination, critically relies on the choice of the lipids that form the LCP around room temperature[1,2]. Owing to the regularly branched chain structure, IPCL-LCPs are characterized by the low LC → LCP phase transition temperature (TK): most of the TK values are close to or below 0 °C, as shown in Supplementary Table 110–14. They are attractive as a new crystallization matrix lipid for temperature-sensitive proteins and as an LC phase formation-free crystal delivery medium in LCP-SFX. The utility of IPCLs as a possible crystal delivery medium in LCP-SFX has not been evaluated

Methods

Results

Conclusion