Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland.
Methods Mol Biol. 2021;2215:267-284. doi: 10.1007/978-1-0716-0966-8_12.
Electron crystallography has been used for decades to determine three-dimensional structures of membrane proteins embedded in a lipid bilayer. However, high-resolution information could only be retrieved from samples where the 2D crystals were well ordered and perfectly flat. This is rarely the case in practice. We implemented in the FOCUS package a module to export transmission electron microscopy images of 2D crystals for 3D reconstruction by single particle algorithms. This approach allows for correcting local distortions of the 2D crystals, yielding much higher resolution reconstructions than otherwise expected from the observable diffraction spots. In addition, the single particle framework enables classification of heterogeneous structures coexisting within the 2D crystals. We provide here a detailed guide on single particle analysis of 2D crystal data based on the FOCUS and FREALIGN packages.
电子晶体学已经被用于确定嵌入在脂质双层中的膜蛋白的三维结构数十年。然而，只有在 2D 晶体排列整齐且完全平整的情况下，才能获得高分辨率的信息。在实际中，这种情况很少见。我们在 FOCUS 软件包中实现了一个模块，可以导出 2D 晶体的透射电子显微镜图像，以便通过单颗粒算法进行 3D 重建。这种方法可以纠正 2D 晶体的局部变形，从而获得比仅从可观察到的衍射点预期的分辨率更高的重建结果。此外，单颗粒框架还可以对 2D 晶体中共存的异构结构进行分类。我们在此提供了一个基于 FOCUS 和 FREALIGN 软件包的详细指南，介绍如何对 2D 晶体数据进行单颗粒分析。
