Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Institute for Protein Research, Osaka University, Osaka, Japan.
Methods Mol Biol. 2020;2134:131-138. doi: 10.1007/978-1-0716-0459-5_12.
Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is capable of determining protein structures at resolutions high enough to visualize their individual domains and secondary structures in their cellular environment.
螺旋体，如密螺旋体、伯氏疏螺旋体和钩端螺旋体，能够通过旋转周质鞭毛或内生鞭毛在液体环境中游泳，这些鞭毛存在于细胞内部。电子冷冻断层扫描（ECT）是一种成像技术，可直接提供纳米分辨率下细胞及其细胞环境中分子复合物的三维（3D）结构。在这里，我提出了一个一般的 ECT 方案，涵盖了样品制备、数据收集、倾斜系列对齐和断层重建，用于可视化钩端螺旋体中完整的周质鞭毛。该方案能够确定分辨率足够高的蛋白质结构，以可视化其在细胞环境中的各个结构域和二级结构。
