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Through-grid wicking enables high-speed cryoEM specimen preparation

View ORCID ProfileYong Zi Tan, View ORCID ProfileJohn L. Rubinstein
doi: https://doi.org/10.1101/2020.05.03.075366
Yong Zi Tan
1Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada
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John L. Rubinstein
1Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada
2Department of Biochemistry, The University of Toronto, Ontario, Canada
3Department of Medical Biophysics, The University of Toronto, Ontario, Canada
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  • For correspondence: john.rubinstein@utoronto.ca
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Abstract

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. We show that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass fiber filter held against the opposite side, often called the ‘back’ of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. We combined ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device that we name ‘Back-it-up’, or BIU. The high liquid-absorption capacity of the glass fiber compared to self-wicking grids appears to make the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The device’s speed increases the number of views for a specimen that suffers from preferred orientations.

Competing Interest Statement

YZT and JLR have filed a patent application related to the specimen preparation approach described in this manuscript.

Footnotes

  • New data has been added that demonstrates that through-grid wicking may be used with membrane protein complexes in detergent.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 16, 2020.
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Through-grid wicking enables high-speed cryoEM specimen preparation
Yong Zi Tan, John L. Rubinstein
bioRxiv 2020.05.03.075366; doi: https://doi.org/10.1101/2020.05.03.075366
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Through-grid wicking enables high-speed cryoEM specimen preparation
Yong Zi Tan, John L. Rubinstein
bioRxiv 2020.05.03.075366; doi: https://doi.org/10.1101/2020.05.03.075366

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