Kontziampasis, D orcid.org/0000-0002-6787-8892, Klebl, DP, Iadanza, MG et al. (7 more authors) (2019) A cryo-EM grid preparation device for time-resolved structural studies. IUCrJ, 6 (6). ISSN 2052-2525
Abstract
Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | This article is protected by copyright. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. |
Keywords: | time-resolved cryo-EM; sample preparation; microscope hardware; structural biology; voltage-assisted spraying |
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) > Animal Cell Biology (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Funding Information: | Funder Grant number BBSRC BB/P026397/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 08 Aug 2019 10:33 |
Last Modified: | 09 Sep 2019 12:47 |
Status: | Published |
Publisher: | International Union of Crystallography |
Identification Number: | 10.1107/S2052252519011345 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:149427 |