Tan Yong Zi, Rubinstein John L
Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada.
Acta Crystallogr D Struct Biol. 2020 Nov 1;76(Pt 11):1092-1103. doi: 10.1107/S2059798320012474. Epub 2020 Oct 13.
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. Here, it is shown 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. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
传统冷冻电镜样本制备的印迹时间使时间分辨研究变得复杂，并导致一些样本在空气-水界面处呈现出优先取向或变性。本文表明，喷在有孔冷冻电镜网格一侧的溶液可以通过贴在网格另一侧（通常称为“背面”）的玻璃纤维滤纸吸过网格，从而形成适合玻璃化的薄膜。这个过程可以在几十毫秒内完成。超声样本施加和穿网吸液技术被整合到一个名为“Back-it-up”或BIU的高速样本制备装置中。与自吸液网格相比，玻璃纤维的高液体吸收能力使得该方法对所施加样本的量相对不敏感。因此，穿网吸液会产生大面积适合冷冻电镜观察的冰，适用于可溶性和去污剂增溶的蛋白质复合物。该装置的速度增加了对于存在优先取向问题的样本的观察次数。
