High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

Abstract

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.

Key words

Electron cryo-tomography Subtomogram averaging Molecular machines Protein self-assembly Structural biology 
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Notes

Acknowledgment

LH was supported by a Biotechnology and Biological Sciences Research Council postgraduate training award and Biotechnology and Biological Sciences Research Council Grant BB/L023091/1 to MB.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life SciencesImperial College of LondonLondonUK

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