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

TitleCryo-EM analysis of a membrane protein embedded in the liposome.
Publication TypeJournal Article
Year of Publication2020
AuthorsYao, X, Fan, X, Yan, N
JournalProc Natl Acad Sci U S A
Volume117
Issue31
Pagination18497-18503
Date Published2020 08 04
ISSN1091-6490
KeywordsCell Membrane, Cryoelectron Microscopy, Escherichia coli, Escherichia coli Proteins, Liposomes, Membrane Proteins, Models, Molecular, Multidrug Resistance-Associated Proteins, Protein Conformation
Abstract

<p>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.</p>

DOI10.1073/pnas.2009385117
Alternate JournalProc Natl Acad Sci U S A
PubMed ID32680969
PubMed Central IDPMC7414195
Grant ListR01 GM130762 / GM / NIGMS NIH HHS / United States