Zhu Shiwei, Qin Zhuan, Wang Juyu, Morado Dustin R, Liu Jun
Department of Pathology and Laboratory Medicine, McGovern Medical School at UTHealth, 6431 Fannin Street, MSB 2.228, Houston, TX, 77030, USA.
Methods Mol Biol. 2017;1593:229-242. doi: 10.1007/978-1-4939-6927-2_18.
The bacterial flagellar motor is a large multi-component molecular machine. Structural determination of such a large complex is often challenging and requires extensive structural analysis in situ. Cryo-electron tomography (cryo-ET) has emerged as a powerful technique that enables us to visualize intact flagellar motors in cells with unprecedented details. Here, we detail the procedure beginning with sample preparation, followed by data acquisition, tomographic reconstruction, sub-tomogram analysis, and ultimately visualization of the intact spirochetal flagellar motor in Borrelia burgdorferi. The procedure is applicable to visualize other molecular machinery in bacteria or other organisms.
细菌鞭毛马达是一种大型多组分分子机器。确定如此大型复合物的结构通常具有挑战性，需要进行广泛的原位结构分析。冷冻电子断层扫描（cryo-ET）已成为一种强大的技术，使我们能够以前所未有的细节可视化细胞中的完整鞭毛马达。在这里，我们详细介绍了从样品制备开始的过程，随后是数据采集、断层重建、亚断层分析，最终可视化伯氏疏螺旋体中完整的螺旋体鞭毛马达。该过程适用于可视化细菌或其他生物体中的其他分子机器。
