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In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography

Kaplan, Mohammed and Nicolas, William J. and Zhao, Wei and Carter, Stephen D. and Metskas, Lauren Ann and Chreifi, Georges and Ghosal, Debnath and Jensen, Grant J. (2020) In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography. In: cryoEM: Methods and Protocols. Methods in Molecular Biology. No.2215. Humana Press , New York, NY, pp. 83-111. ISBN 978-1-0716-0965-1. https://resolver.caltech.edu/CaltechAUTHORS:20210111-153220027

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Abstract

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).


Item Type:Book Section
Related URLs:
URLURL TypeDescription
https://doi.org/10.1007/978-1-0716-0966-8_4DOIArticle
ORCID:
AuthorORCID
Kaplan, Mohammed0000-0002-0759-0459
Nicolas, William J.0000-0001-5970-8626
Carter, Stephen D.0000-0002-4237-4276
Metskas, Lauren Ann0000-0002-8073-6960
Chreifi, Georges0000-0003-4194-1694
Ghosal, Debnath0000-0002-2227-0330
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© Springer Science+Business Media, LLC, part of Springer Nature 2021. First Online: 27 December 2020. Cryo-ET work in the Jensen lab is supported by NIH grants R35 GM122588, RO1 A127401, and P50 AI150464 and by the Howard Hughes Medical Institute and performed in the Beckman Institute Resource Center for Transmission Electron Microscopy. M.K. acknowledges a Rubicon postdoctoral fellowship from De Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). We thank Catherine M Oikonomou for reading and editing the manuscript.
Funders:
Funding AgencyGrant Number
NIHR35 GM122588
NIHRO1 A127401
NIHP50 AI150464
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)UNSPECIFIED
Subject Keywords:Cryo-ET; In situ structural biology; Subtomogram averaging; FIB-milling; High-resolution tomography; Cryosectioning
Series Name:Methods in Molecular Biology
Issue or Number:2215
Record Number:CaltechAUTHORS:20210111-153220027
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210111-153220027
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:107398
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Jan 2021 23:50
Last Modified:11 Jan 2021 23:50

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