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Legionella pp 249-265 | Cite as

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

  • Debnath Ghosal
  • Mohammed Kaplan
  • Yi-Wei Chang
  • Grant J. JensenEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Bacterial secretion system Legionella Dot/Icm T4SS Electron cryotomography (ECT) Subtomogram averaging 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Debnath Ghosal
    • 1
  • Mohammed Kaplan
    • 1
  • Yi-Wei Chang
    • 1
  • Grant J. Jensen
    • 1
    • 2
    Email author
  1. 1.Division of Biology and Biological EngineeringCalifornia Institute of TechnologyPasadenaUSA
  2. 2.Howard Hughes Medical InstitutePasadenaUSA

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