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

Ghosal, Debnath and Kaplan, Mohammed and Chang, Yi-Wei and Jensen, Grant J. (2019) In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography. In: Legionella. Methods in Molecular Biology. No.1921. Humana Press , New York, NY, pp. 249-265. ISBN 978-1-4939-9047-4. https://resolver.caltech.edu/CaltechAUTHORS:20190211-094654581

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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.


Item Type:Book Section
Related URLs:
URLURL TypeDescription
https://doi.org/10.1007/978-1-4939-9048-1_16DOIArticle
ORCID:
AuthorORCID
Ghosal, Debnath0000-0002-2227-0330
Kaplan, Mohammed0000-0002-0759-0459
Chang, Yi-Wei0000-0003-2391-473X
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2019 Springer Science+Business Media, LLC, part of Springer Nature. First Online: 30 January 2019.
Subject Keywords:Bacterial secretion system; Legionella Dot/Icm T4SS; Electron cryotomography (ECT); Subtomogram averaging
Series Name:Methods in Molecular Biology
Issue or Number:1921
Record Number:CaltechAUTHORS:20190211-094654581
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190211-094654581
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:92827
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Feb 2019 21:42
Last Modified:03 Oct 2019 20:48

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