Year: 2022
Type: article
Abstract: Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although the similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which led to structures of antagonist bound A 2A adenosine receptor at 3.4Å resolution and unliganded Smoothened at 3.7Å resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.
Source: Nature Communications
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $5,890
Funders: Howard Hughes Medical Institute, Pew Charitable Trusts, Vallee Foundation, Edward Mallinckrodt, Jr. Foundation, Searle Scholars Program, Human Frontier Science Program, National Institutes of Health, University of California, San Francisco, National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences
Awards: Molecular Biophysics Training Grant, Glacios™ Cryo Transmission Electron Microscope with 200 kV XFEG optics, Acquisition of an electron microscope for high-resolution single particle cryo-EM, Biochemical approaches to enlighten GPR85 function, G7383342067, Molecular mechanisms of proton-sensing in CO2-dependent breathing, Linux cluster for near atomic resolution single particle cryo-EM, G8873047370, Mechanisms of Smoothened Activation in Hedgehog Signaling, Advancing cryo-EM technology to address difficult biological questions
OpenAlex ID: W4288077437