Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China.
Ministry of Education Key Laboratory of Protein Sciences, Beijing Frontier Research Center for Biological Structures, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Nat Commun. 2024 Aug 24;15(1):7300. doi: 10.1038/s41467-024-51696-w.
Cryo-electron microscopy (cryo-EM) has been widely used to reveal the structures of proteins at atomic resolution. One key challenge is that almost all proteins are predominantly adsorbed to the air-water interface during standard cryo-EM specimen preparation. The interaction of proteins with air-water interface will significantly impede the success of reconstruction and achievable resolution. Here, we highlight the critical role of impenetrable surfactant monolayers in passivating the air-water interface problems, and develop a robust effective method for high-resolution cryo-EM analysis, by using the superstructure GSAMs which comprises surfactant self-assembled monolayers (SAMs) and graphene membrane. The GSAMs works well in enriching the orientations and improving particle utilization ratio of multiple proteins, facilitating the 3.3-Å resolution reconstruction of a 100-kDa protein complex (ACE2-RBD), which shows strong preferential orientation using traditional specimen preparation protocol. Additionally, we demonstrate that GSAMs enables the successful determinations of small proteins (<100 kDa) at near-atomic resolution. This study expands the understanding of SAMs and provides a key to better control the interaction of protein with air-water interface.
冷冻电子显微镜（cryo-EM）已广泛用于以原子分辨率揭示蛋白质的结构。一个关键的挑战是，在标准的冷冻电子显微镜标本制备过程中，几乎所有的蛋白质主要都被吸附在气-水界面上。蛋白质与气-水界面的相互作用会严重阻碍重构的成功和可达到的分辨率。在这里，我们强调了不可渗透的表面活性剂单层在钝化气-水界面问题方面的关键作用，并通过使用由表面活性剂自组装单层（SAMs）和石墨烯膜组成的超结构 GSAMs，开发了一种用于高分辨率冷冻电子显微镜分析的稳健有效方法。GSAMs 可以很好地富集多个蛋白质的取向并提高粒子利用率，促进了 ACE2-RBD 这种 100kDa 蛋白复合物以 3.3Å 的分辨率进行重构，这表明使用传统的标本制备方案具有很强的优先取向。此外，我们证明 GSAMs 可以成功地以近原子分辨率确定小于 100kDa 的小蛋白。这项研究扩展了对 SAMs 的理解，并为更好地控制蛋白质与气-水界面的相互作用提供了关键。
