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

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

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.

Key words

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

Notes

Acknowledgments

Wellcome Trust and Human Frontier Science Program provided funding for this work. The work was also supported by NIH grant NIH 5P41GM103832.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of CambridgeCambridgeUK
  2. 2.National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular BiologyBaylor College of MedicineHoustonUSA

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