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

Michael W. Martynowycz, Anna Shiriaeva, Xuanrui Ge, Johan Hattne, Brent L. Nannenga, Vadim Cherezov, Tamir Gonen

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbere2106041118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number36
DOIs
StatePublished - Sep 7 2021
Externally publishedYes

Keywords

  • GPCR
  • Ion-beam milling
  • Lipidic cubic phase
  • Membrane proteins
  • MicroED

ASJC Scopus subject areas

  • General

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