National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS), Beijing 100101, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
J Struct Biol. 2021 Dec;213(4):107783. doi: 10.1016/j.jsb.2021.107783. Epub 2021 Aug 25.
The air-water interface (AWI) tends to adsorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, some of the nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. Our results indicate that proteins adsorb to charged interface and the negative charge of the AWI plays an important role in adsorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed by adding anionic detergent at a concentration between 0.002% and 0.005%. Using this method, the protein particles exhibited a more evenly distributed orientations and still adsorbed to the AWI enabling them embedding in a thin layer of ice with high concentration, which will benefit the cryo-EM structural determination.
气-液界面（AWI）倾向于吸附蛋白质，并且经常在低温电子显微镜（cryo-EM）中引起优先取向问题。在这里，我们检查了用不同去污剂冷冻的蛋白质样品的低温电子显微镜数据，发现阴离子和阳离子去污剂都促进了蛋白质与 AWI 的结合。相比之下，一些非离子型和两性离子型去污剂往往会阻止蛋白质附着在 AWI 上。不同阴离子去污剂的蛋白质取向分布相似，与无去污剂时获得的结果相似。相比之下，阳离子去污剂给出了不同的取向分布。我们的结果表明，蛋白质吸附在带电荷的界面上，而 AWI 的负电荷在传统低温电子显微镜样品制备中吸附蛋白质中起着重要作用。根据这些发现，开发了一种新方法，即在浓度为 0.002%和 0.005%之间添加阴离子去污剂。使用这种方法，蛋白质颗粒表现出更均匀分布的取向，并且仍然吸附在 AWI 上，使它们能够嵌入高浓度的薄冰层中，这将有利于低温电子显微镜结构测定。
