Purification of AcrAB-TolC Multidrug Efflux Pump for Cryo-EM Analysis

Methods Mol Biol. 2018:1700:71-81. doi: 10.1007/978-1-4939-7454-2_5.

Abstract

The cell envelope of Gram-negative bacteria comprises an outer membrane, a cytoplasmic inner membrane, and an interstitial space. The tripartite multidrug transporter AcrAB-TolC, which uses proton electrochemical gradients to vectorially drive the efflux of drugs from the cell, spans this envelope. We describe here details of the methods used to prepare the recombinant tripartite assembly for high-resolution structure determination by cryo-EM.

Keywords: Cryo-EM; Inner membrane; Outer membrane protein; RND family; Three-dimensional structure; Transmembrane protein assembly; Transport mechanism.

MeSH terms

  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / isolation & purification*
  • Cryoelectron Microscopy
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification*
  • Models, Molecular
  • Mutation
  • Protein Conformation

Substances

  • AcrAB-TolC protein, E coli
  • Carrier Proteins
  • Escherichia coli Proteins