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Fusion protein strategies for cryo-EM study of G protein-coupled receptors
Open accessPreprint — published as 10.1038/s41467-022-32125-2 · Paper page
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.
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Highly accurate protein structure prediction with AlphaFold
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Metadata
- Authors
- Zhang, K., Wu, H., Hoppe, N., Manglik, A., & Cheng, Y.
- Affiliations
- University of California, San Francisco (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Manglik, Aashish
- Cheng, Yifan
- Venue
- Nature Communications
- Publisher
- openRxiv
- Published
- 2021-12-24
- Volume
- 13
- Issue
- 1
- Pages
- 4366-4366
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC9334595
- OpenAlex
- W4288077437
- Cited by
- 91
- References
- 1
- Citations by year
- 2026: 22 · 2025: 28 · 2024: 20 · 2023: 18 · 2022: 3
- FWCI
- 5.63
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Nature Communications (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- 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 (1s10od026881-01, 1s10od021741-01, 1r03tr003384-01, 1s10od020054-01, S10OD025881)
- University of California, San Francisco (R35GM140847)
- National Heart, Lung, and Blood Institute (F31HL164045)
- National Institute of General Medical Sciences (T32GM008284, R01GM138992, R35GM140847)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
- Keywords
- G protein-coupled receptors, fusion protein, single-particle cryo-EM, adenosine A2A receptor, Smoothened, unliganded receptor, integral membrane proteins, image registration
- MeSH terms
- Crystallography, Protein Structure, Secondary, GTP-Binding Proteins, Cryoelectron Microscopy, Receptors, G-Protein-Coupled
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