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Bacterial Chemosensing pp 187–199Cite as

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

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1729))

Abstract

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.

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References

  1. Briggs JA (2013) Structural biology in situ—the potential of subtomogram averaging. Curr Opin Struct Biol 23:261–267

    Article  CAS  Google Scholar 

  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 165:64–77

    Article  CAS  Google Scholar 

  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 106:17181–17186

    Article  CAS  Google Scholar 

  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 109:3766–3771

    Article  CAS  Google Scholar 

  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–E1488

    Article  CAS  Google Scholar 

  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 127:11709–11719

    Article  CAS  Google Scholar 

  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:e08419

    Article  Google Scholar 

  8. Li M, Hazelbauer GL (2011) Core unit of chemotaxis signaling complexes. Proc Natl Acad Sci U S A 108:9390–9395

    Article  CAS  Google Scholar 

  9. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

    Article  Google Scholar 

  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 10:584–590

    Article  CAS  Google Scholar 

  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 25:1605–1612

    Article  CAS  Google Scholar 

  12. Morado DR, Hu B, Liu J (2016) Using Tomoauto: a protocol for high-throughput automated cryo-electron tomography. J Vis Exp 107:e53608

    Google Scholar 

  13. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76

    Article  CAS  Google Scholar 

  14. Agulleiro JI, Fernandez JJ (2015) Tomo3D 2.0—exploitation of advanced vector extensions (AVX) for 3D reconstruction. J Struct Biol 189:147–152

    Article  Google Scholar 

  15. Winkler H (2007) 3D reconstruction and processing of volumetric data in cryo-electron tomography. J Struct Biol 157:126–137

    Article  CAS  Google Scholar 

  16. Goddard TD, Huang CC, Ferrin TE (2007) Visualizing density maps with UCSF Chimera. J Struct Biol 157:281–287

    Article  CAS  Google Scholar 

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Acknowledgment

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.

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Authors and Affiliations

  1. Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale School of Medicine, Advanced Biosciences Center, New Haven, CT, USA

    Zhuan Qin & Jun Liu

  2. Department of Microbiology and Molecular Biology, McGovern Medical School, The University of Texas, Houston, TX, USA

    Bo Hu

Authors
  1. Zhuan Qin
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  2. Bo Hu
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  3. Jun Liu
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Corresponding author

Correspondence to Jun Liu .

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Editors and Affiliations

  1. Department of Biology, Texas A&M University, College Station, Texas, USA

    Michael D. Manson

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Qin, Z., Hu, B., Liu, J. (2018). Visualizing Chemoreceptor Arrays in Bacterial Minicells by Cryo-Electron Tomography and Subtomogram Analysis. In: Manson, M. (eds) Bacterial Chemosensing. Methods in Molecular Biology, vol 1729. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7577-8_17

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  • DOI: https://doi.org/10.1007/978-1-4939-7577-8_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7576-1

  • Online ISBN: 978-1-4939-7577-8

  • eBook Packages: Springer Protocols

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