RT Journal Article
SR Electronic
T1 Through-grid wicking enables high-speed cryoEM specimen preparation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.05.03.075366
DO 10.1101/2020.05.03.075366
A1 Tan, Yong Zi
A1 Rubinstein, John L.
YR 2020
UL http://biorxiv.org/content/early/2020/07/16/2020.05.03.075366.abstract
AB 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 StatementYZT and JLR have filed a patent application related to the specimen preparation approach described in this manuscript.