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Legionella pp 249–265Cite as

In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1921))

Abstract

Determining the three-dimensional structure of biomacromolecules at high resolution in their native cellular environment is a major challenge for structural biology. Toward this end, electron cryotomography (ECT) allows large bio-macromolecular assemblies to be imaged directly in their hydrated physiological milieu to ~4 nm resolution. Combining ECT with other techniques like fluorescent imaging, immunogold labeling, and genetic manipulation has allowed the in situ investigation of complex biological processes at macromolecular resolution. Furthermore, the advent of cryogenic focused ion beam (FIB) milling has extended the domain of ECT to include regions even deep within thick eukaryotic cells. Anticipating two audiences (scientists who just want to understand the potential and general workflow involved and scientists who are learning how to do the work themselves), here we present both a broad overview of this kind of work and a step-by-step example protocol for ECT and subtomogram averaging using the Legionella pneumophila Dot/Icm type IV secretion system (T4SS) as a case study. While the general workflow is presented in step-by-step detail, we refer to online tutorials, user’s manuals, and other training materials for the essential background understanding needed to perform each step.

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Acknowledgments

We thank Dr. Songye Chen (Caltech). This work is supported by NIH grant R01482AI127401 to G.J.J.

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Author notes

  1. Debnath Ghosal and Mohammed Kaplan have contributed equally to this work

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Debnath Ghosal, Mohammed Kaplan, Yi-Wei Chang & Grant J. Jensen

  2. Howard Hughes Medical Institute, Pasadena, CA, USA

    Grant J. Jensen

Authors
  1. Debnath Ghosal
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  2. Mohammed Kaplan
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  3. Yi-Wei Chang
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  4. Grant J. Jensen
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Corresponding author

Correspondence to Grant J. Jensen .

Editor information

Editors and Affiliations

  1. Institut Pasteur, Biologie des Bactéries Intracellulaires, Paris, France

    Carmen Buchrieser

  2. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland

    Hubert Hilbi

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Ghosal, D., Kaplan, M., Chang, YW., Jensen, G.J. (2019). In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 1921. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9048-1_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9048-1_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9047-4

  • Online ISBN: 978-1-4939-9048-1

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