Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Anna Higgins, Alex Flynn, Anaïs Marconnet, Laura Musgrove, Vincent Postis, Jonathan Lippiat, Chun-Wa Chung, Tom Ceska, Manuela Zoonens, Frank Sobott, Stephen Muench

Research output: Other contribution

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Abstract

Membrane proteins are essential for cellular growth and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates significant challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a significant advantage. Here we use a novel type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful detergent-free approach for the study of membrane proteins allowing native extraction and high-resolution structure determination by cryoEM.
Original languageEnglish
TypePreprint
Media of outputResearch Square
PublisherResearch Square
DOIs
Publication statusPublished - Jan 2021

Keywords

  • CryoEM
  • membrane protein
  • amphipol
  • detergent

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