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

Xia Yao, Xiao Fan, Nieng Yan

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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 threedimensional (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.

Original languageEnglish (US)
Pages (from-to)18497-18503
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number31
DOIs
StatePublished - Aug 4 2020

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Cryo-em
  • Graphene grids
  • Membrane protein
  • Proteoliposome
  • Structural biology

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