Laboratory of Genetics, The Salk Institute for Biological Studies.
Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies.
J Vis Exp. 2022 Jul 1(185). doi: 10.3791/64136.
Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.
冷冻电子显微镜（cryo-EM）的单颗粒分析（SPA）现在是高分辨率结构生物学的主流技术。通过 SPA 进行结构测定依赖于获得在薄冰层中冷冻的大分子物体的多个不同视角。理想情况下，一组均匀分布的随机投影方向将构成物体的所有可能视角，从而产生具有各向同性方向分辨率的重建。然而，在现实中，许多样品都存在优先取向的粒子附着在气-水界面上的问题。这会导致数据集的角向分布不均匀，以及重建中的傅立叶空间采样不均匀，从而导致具有各向异性分辨率的图谱。通过倾斜样品台，可以通过提高方向分布的均匀性来普遍克服分辨率各向异性，从而改善傅立叶空间采样的各向同性。本方案描述了一种使用 Leginon（一种用于自动化图像采集的软件）的倾斜台自动数据采集策略。该程序易于实施，不需要任何额外的设备或软件，并且与用于成像生物大分子的大多数标准透射电子显微镜（TEM）兼容。
