Cryo-EM analysis of a membrane protein embedded in the liposome.

Szczegóły
Abstrakt

Tytuł:: Cryo-EM analysis of a membrane protein embedded in the liposome.
Autorzy:: Yao X; Department of Molecular Biology, Princeton University, Princeton, NJ 08544.
Fan X; Department of Molecular Biology, Princeton University, Princeton, NJ 08544 .
Yan N; Department of Molecular Biology, Princeton University, Princeton, NJ 08544 .
Źródło:: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Aug 04; Vol. 117 (31), pp. 18497-18503. Date of Electronic Publication: 2020 Jul 17.
Typ publikacji:: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.
Język:: English
Imprint Name(s):: Original Publication: Washington, DC : National Academy of Sciences
MeSH Terms:: Escherichia coli/*metabolism
Escherichia coli Proteins/*metabolism
Liposomes/*ultrastructure
Membrane Proteins/*metabolism
Multidrug Resistance-Associated Proteins/*metabolism
Cell Membrane/metabolism ; Cell Membrane/ultrastructure ; Cryoelectron Microscopy ; Escherichia coli/ultrastructure ; Escherichia coli Proteins/ultrastructure ; Liposomes/metabolism ; Membrane Proteins/ultrastructure ; Models, Molecular ; Multidrug Resistance-Associated Proteins/ultrastructure ; Protein Conformation
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Grant Information:: R01 GM130762 United States GM NIGMS NIH HHS
Contributed Indexing:: Keywords: cryo-EM; graphene grids; membrane protein; proteoliposome; structural biology
Substance Nomenclature:: 0 (AcrB protein, E coli)
0 (Escherichia coli Proteins)
0 (Liposomes)
0 (Membrane Proteins)
0 (Multidrug Resistance-Associated Proteins)
Entry Date(s):: Date Created: 20200719 Date Completed: 20200930 Latest Revision: 20240329
Update Code:: 20240329
PubMed Central ID:: PMC7414195
DOI:: 10.1073/pnas.2009385117
PMID:: 32680969
: Czasopismo naukowe

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here we present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 Å resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Our approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.
Competing Interests: The authors declare no competing interest.

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