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.

空气-水界面 (AWI) 倾向于吸附蛋白质，并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冷冻电镜数据，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，一些非离子和两性离子去污剂倾向于阻止蛋白质附着在 AWI 上。使用不同阴离子去污剂的蛋白质取向分布是相似的，并且类似于没有去污剂的情况。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，蛋白质吸附在带电界面上，AWI 的负电荷在常规冷冻电镜样品制备中吸附蛋白质方面起着重要作用。根据这些发现，开发了一种新方法，即添加浓度在 0.002% 和 0.005% 之间的阴离子去污剂。使用这种方法，蛋白质颗粒表现出更均匀的分布方向，并且仍然吸附在 AWI 上，使其能够嵌入到高浓度的薄冰层中，这将有利于低温电镜结构的测定。