Klebl, DP, Sobott, F orcid.org/0000-0001-9029-1865, White, HD et al. (1 more author) (2021) Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy. Journal of Visualized Experiments, 177. e62199. ISSN 1940-087X
Abstract
The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 µL sample at concentrations of ≥ 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.
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 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: | 01 Sep 2021 15:39 |
Last Modified: | 18 Nov 2024 13:56 |
Status: | Published |
Publisher: | MyJove Corporation |
Identification Number: | 10.3791/62199 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:177615 |