White, JBR, Maskell, DP, Howe, A et al. (5 more authors) (2021) Single Particle Cryo-Electron Microscopy: From Sample to Structure. Journal of Visualized Experiments (171). e62415. ISSN 1940-087X
Abstract
Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 JoVE Journal of Visualized Experiments. This is an author produced version of an article, published in Journal of Visualized Experiments. Uploaded in accordance with the publisher's self-archiving policy. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 28 Jun 2021 09:51 |
Last Modified: | 29 Nov 2021 01:38 |
Status: | Published |
Publisher: | MyJove Corporation |
Identification Number: | 10.3791/62415 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:175632 |