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

Sci Rep. 2020 Nov 9;10(1):19305. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Lipids
  • Membrane Proteins
  • Monoglycerides
  • Receptors, Adenosine A2
  • Terpenes
  • Cholesterol