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 language | English (US) |
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Article number | e2106041118 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 118 |
Issue number | 36 |
DOIs | |
State | Published - Sep 7 2021 |
Keywords
- GPCR
- Ion-beam milling
- Lipidic cubic phase
- Membrane proteins
- MicroED
ASJC Scopus subject areas
- General
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MicroED structure of the human adenosine receptor at 2.8A
Martynowycz, M. W. (Contributor), Shiriaeva, A. (Contributor), Ge, X. (Contributor), Hattne, J. (Contributor), Nannenga, B. (Contributor), Cherezov, V. (Contributor) & Gonen, T. (Contributor), Protein Data Bank (PDB), Sep 8 2021
DOI: 10.2210/pdb7RM5, https://www.wwpdb.org/pdb?id=pdb_00007rm5
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