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 EROCOC(17+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 A(2A) receptor (A(2A)R). The structures of EROCOC(17+4)-matrix grown A(2A)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(2A)R crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC(17+4)/water system did not form the crystalline L(C) phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline L(C) phase below about 17 °C. As the L(C) phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC(17+4) will expand the utility of the in meso method.