Through-grid wicking enables high-speed cryoEM specimen preparation
Through-grid wicking enables high-speed cryoEM specimen preparation
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DOI：
10.1107/s2059798320012474
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发表时间：
2020-11-01
影响因子：
2.2
通讯作者：
Rubinstein, John L.
中科院分区：
文献类型：
--
作者：
Tan, Yong Zi;Rubinstein, John L.
常规cryoEM标本制备的印迹时间使时间分辨研究复杂化，并导致一些标本在空气-水界面处采用优选取向或变性。在这里，它表明，溶液喷雾到一侧的多孔cryoEM网格可以芯吸通过网格的玻璃纤维过滤器保持对相对侧，通常被称为“回”，网格，产生适合于玻璃化的膜。这个过程可以在几十毫秒内完成。超声波样品应用和通过网格芯吸结合在一个高速的微波制备装置，被命名为“备份”或BIU。与自芯吸滤线栅相比，玻璃纤维的高液体吸收能力使得该方法对所施加的样品量相对不敏感。因此，通过网格芯吸产生大面积的冰，其适合用于可溶性和洗涤剂溶解的蛋白质复合物的cryoEM。设备的速度增加了受优选取向影响的样本的视图数量。
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.