Visualizing Chemoreceptor Arrays in Bacterial Minicells by Cryo-Electron Tomography and Subtomogram Analysis

  • Zhuan Qin
  • Bo Hu
  • Jun Liu
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)


Bacterial chemoreceptors form a highly ordered array in concert with the CheA kinase and the CheW coupling protein. The precise architecture of the array is responsible for high sensitivity, high dynamic range, and strong amplification of chemotaxis signaling. Cryo-electron tomography (cryo-ET) has emerged as a unique tool to visualize bacterial chemotaxis arrays at molecular level. Here we describe a detailed cryo-ET and subtomogram averaging procedure to determine in situ structure of the chemoreceptor arrays in Salmonella minicells. The procedure should be readily applicable to visualize other large macromolecular assemblies in their cellular context.


Cryo-electron tomography Chemoreceptor array Core signaling complex Subtomogram averaging Minicell Macromolecular assembly 



This work was supported in part by grants R01AI087946 from the NIAID, R01GM110243 from the NIGMS, and AU-1714 from the Welch Foundation. The direct electron detector was funded by NIH award S10OD016279.


  1. 1.
    Briggs JA (2013) Structural biology in situ—the potential of subtomogram averaging. Curr Opin Struct Biol 23:261–267CrossRefGoogle Scholar
  2. 2.
    Winkler H, Zhu P, Liu J, Ye F, Roux KH et al (2009) Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes. J Struct Biol 165:64–77CrossRefGoogle Scholar
  3. 3.
    Briegel A, Ortega DR, Tocheva EI, Wuichet K, Li Z et al (2009) Universal architecture of bacterial chemoreceptor arrays. Proc Natl Acad Sci U S A 106:17181–17186CrossRefGoogle Scholar
  4. 4.
    Briegel A, Li X, Bilwes AM, Hughes KT, Jensen GJ et al (2012) Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins. Proc Natl Acad Sci U S A 109:3766–3771CrossRefGoogle Scholar
  5. 5.
    Liu J, Hu B, Morado DR, Jani S, Manson MD et al (2012) Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells. Proc Natl Acad Sci U S A 109:E1481–E1488CrossRefGoogle Scholar
  6. 6.
    Zhang J, Xu Y, Shen J, Luo X, Chen J et al (2005) Dynamic mechanism for the autophosphorylation of CheA histidine kinase: molecular dynamics simulations. J Am Chem Soc 127:11709–11719CrossRefGoogle Scholar
  7. 7.
    Cassidy CK, Himes BA, Alvarez FJ, Ma J, Zhao G et al (2015) CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling. Elife 4:pii:e08419CrossRefGoogle Scholar
  8. 8.
    Li M, Hazelbauer GL (2011) Core unit of chemotaxis signaling complexes. Proc Natl Acad Sci U S A 108:9390–9395CrossRefGoogle Scholar
  9. 9.
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51CrossRefGoogle Scholar
  10. 10.
    Li X, Mooney P, Zheng S, Booth CR, Braunfeld MB et al (2013) Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat Methods 10:584–590CrossRefGoogle Scholar
  11. 11.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  12. 12.
    Morado DR, Hu B, Liu J (2016) Using Tomoauto: a protocol for high-throughput automated cryo-electron tomography. J Vis Exp 107:e53608Google Scholar
  13. 13.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76CrossRefGoogle Scholar
  14. 14.
    Agulleiro JI, Fernandez JJ (2015) Tomo3D 2.0—exploitation of advanced vector extensions (AVX) for 3D reconstruction. J Struct Biol 189:147–152CrossRefGoogle Scholar
  15. 15.
    Winkler H (2007) 3D reconstruction and processing of volumetric data in cryo-electron tomography. J Struct Biol 157:126–137CrossRefGoogle Scholar
  16. 16.
    Goddard TD, Huang CC, Ferrin TE (2007) Visualizing density maps with UCSF Chimera. J Struct Biol 157:281–287CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Microbial Pathogenesis and Microbial Sciences InstituteYale School of Medicine, Advanced Biosciences CenterNew HavenUSA
  2. 2.Department of Microbiology and Molecular BiologyMcGovern Medical School, The University of TexasHoustonUSA

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