Crystallographic Analysis of the CusBA Heavy-Metal Efflux Complex of Escherichia coli

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

Abstract

Crystallization is one of the most successful techniques used to determine protein structure, especially for membrane proteins. However, the application of this technique is not straightforward and often hampered by the difficulties associated with expression, purification, and crystallization. Here we present our protocol and methodology for crystallizing the CusBA adaptor–transporter complex of Escherichia coli. Using these procedures, we were able to produce the first co-crystal structure of a resistance-nodulation-cell division (RND) transporter in complex with its associated membrane fusion protein.

Key words

Membrane protein X-ray crystallography Vapor diffusion Heavy-metal efflux Antimicrobial resistance 

Notes

Acknowledgements

This work was supported by an NIH grant R01AI114629 to E.W.Y.

References

  1. 1.
    White SH (2015) Membrane proteins of known 3D structure. http://blanco.biomol.uci.edu/mpstruc/
  2. 2.
    Long F, Su CC, Zimmermann MT, Boyken SE, Rajashankar KR, Jernigan RL, Yu EW (2010) Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature 467:484–488CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Su CC, Yang F, Long F, Reyon D, Routh MD, Kuo DW, Mokhtari AK, Van Ornam JD, Rabe KL, Hoy JA, Lee YJ, Rajashankar KR, Yu EW (2009) Crystal structure of the membrane fusion protein CusB from Escherichia coli. J Mol Biol 393:342–355CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lei HT, Bolla JR, Bishop NR, Su CC, Yu EW (2014) Crystal structures of CusC review conformational changes accompanying folding and transmembrane channel formation. J Mol Biol 426:403–411CrossRefPubMedGoogle Scholar
  5. 5.
    Kulathila R, Kulathila R, Indic M, van den Berg B (2011) Crystal structure of Escherichia coli CusC, the outer membrane component of a heavy metal efflux pump. PLoS One 6:e15610CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, Yu EW (2011) Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature 470:558–563CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Su CC, Long F, Lei HT, Bolla JR, Do SV, Rajashankar KR, Yu EW (2012) Charged amino acids (R83, E567, D617, E625, R669, and K678) of CusA are required for metal ion transport in the Cus efflux system. J Mol Biol 422:429–441CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pope B, Kent HM (1996) High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res 43:536–537CrossRefGoogle Scholar
  9. 9.
    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Terwilliger TC (2001) Maximum-likelihood density modification using pattern recognition of structural motifs. Acta Crystallogr D 57:1755–1762CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60:2126–2132CrossRefPubMedGoogle Scholar
  12. 12.
    Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC (2002) PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr 58:1948–1954Google Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Physics and AstronomyIowa State UniversityAmesUSA
  2. 2.Department of ChemistryIowa State UniversityAmesUSA

Personalised recommendations