MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

Proc Natl Acad Sci U S A. 2021 Sep 7;118(36):e2106041118. doi: 10.1073/pnas.2106041118.


G protein-coupled receptors (GPCRs), or seven-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without bound cognate signaling partners, such as a G protein, requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here, we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by focused ion-beam milling. We determined the structure of the A2A adenosine receptor to 2.8-Å resolution and resolved an antagonist in its orthosteric ligand-binding site, as well as four cholesterol molecules bound around the receptor. This study lays the groundwork for future structural studies of lipid-embedded membrane proteins by MicroED using single microcrystals that would be impossible with other crystallographic methods.

Keywords: GPCR; MicroED; ion-beam milling; lipidic cubic phase; membrane proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Humans
  • Lipids / chemistry
  • Nanoparticles / chemistry*
  • Protein Conformation
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, Purinergic P1 / chemistry*


  • Lipids
  • Receptors, G-Protein-Coupled
  • Receptors, Purinergic P1