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On-grid and in-flow mixing for time-resolved cryo-EM
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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
International Union of Crystallography
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489233/ https://www.ncbi.nlm.nih.gov/pubmed/34605427 http://dx.doi.org/10.1107/S2059798321008810 |
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author | Klebl, David P. White, Howard D. Sobott, Frank Muench, Stephen P. |
author_facet | Klebl, David P. White, Howard D. Sobott, Frank Muench, Stephen P. |
author_sort | Klebl, David P. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8489233 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | International Union of Crystallography |
record_format | MEDLINE/PubMed |
spelling | pubmed-84892332021-10-18 On-grid and in-flow mixing for time-resolved cryo-EM Klebl, David P. White, Howard D. Sobott, Frank Muench, Stephen P. Acta Crystallogr D Struct Biol Ccp-EM 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. International Union of Crystallography 2021-09-22 /pmc/articles/PMC8489233/ /pubmed/34605427 http://dx.doi.org/10.1107/S2059798321008810 Text en © David P. Klebl et al. 2021 https://creativecommons.org/licenses/by/4.0/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. |
spellingShingle | Ccp-EM Klebl, David P. White, Howard D. Sobott, Frank Muench, Stephen P. On-grid and in-flow mixing for time-resolved cryo-EM |
title | On-grid and in-flow mixing for time-resolved cryo-EM |
title_full | On-grid and in-flow mixing for time-resolved cryo-EM |
title_fullStr | On-grid and in-flow mixing for time-resolved cryo-EM |
title_full_unstemmed | On-grid and in-flow mixing for time-resolved cryo-EM |
title_short | On-grid and in-flow mixing for time-resolved cryo-EM |
title_sort | on-grid and in-flow mixing for time-resolved cryo-em |
topic | Ccp-EM |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489233/ https://www.ncbi.nlm.nih.gov/pubmed/34605427 http://dx.doi.org/10.1107/S2059798321008810 |
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