Klebl, DP, Gravett, MSC orcid.org/0000-0001-8351-7176, Kontziampasis, D orcid.org/0000-0002-6787-8892 et al. (9 more authors) (2020) Need for speed: Examining protein behavior during cryoEM grid preparation at different timescales. Structure, 28 (11). pp. 1238-1248. ISSN 0969-2126
Abstract
A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
Keywords: | cryoEM; sample preparation; air-water-interface; HSPD1; ribosome |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Funding Information: | Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/P026397/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 31 Jul 2020 12:40 |
Last Modified: | 25 Feb 2025 14:22 |
Status: | Published |
Publisher: | Cell Press |
Identification Number: | 10.1016/j.str.2020.07.018 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:163897 |