Klebl, DP, White, HD, Sobott, F orcid.org/0000-0001-9029-1865 et al. (1 more author) (2021) On-grid and in-flow mixing for time-resolved cryo-EM. Acta Crystallographica Section D: Structural Biology, 77 (10). D77. ISSN 2059-7983
Abstract
Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5–10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7–700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air–water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 David P. Klebl et al. 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: | electron microscopy; time-resolved; myosin; structural biology; protein dynamics |
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) |
Funding Information: | Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/P026397/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 24 Aug 2021 13:51 |
Last Modified: | 17 Dec 2021 12:44 |
Status: | Published |
Publisher: | International Union of Crystallography |
Identification Number: | 10.1107/S2059798321008810 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:177369 |