Kawamoto Akihiro, Namba Keiichi
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Quantitative Biology Center, RIKEN, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Methods Mol Biol. 2017;1593:119-131. doi: 10.1007/978-1-4939-6927-2_9.
The bacterial flagellum is a large assembly of about 30 different proteins and is divided into three parts: filament, hook, and basal body. The machineries for its crucial functions, such as torque generation, rotational switch regulation, protein export, and assembly initiation, are all located around the basal body. Although high-resolution structures of the filament and hook have already been revealed, the structure of the basal body remains elusive. Recently, the purification protocol for the MS ring, which is the core ring of the basal body, has been improved for the structural study of the MS ring by electron cryomicroscopy (cryoEM) and single particle image analysis. The structure of intact basal body has also been revealed in situ at a resolution of a few nanometers by electron cryotomography (ECT) of minicells. Here, we describe the methods for the MS ring purification, Salmonella minicell culture, and cryoEM/ECT data collection and image analysis.
细菌鞭毛是由大约30种不同蛋白质组成的大型组件，分为三部分：鞭毛丝、钩形鞘和基体。其关键功能的机制，如扭矩产生、旋转开关调节、蛋白质输出和组装起始，都位于基体周围。尽管鞭毛丝和钩形鞘的高分辨率结构已经被揭示，但基体的结构仍然难以捉摸。最近，基体核心环——MS环的纯化方案已经得到改进，以便通过电子冷冻显微镜（cryoEM）和单颗粒图像分析对MS环进行结构研究。通过对微小细胞进行电子冷冻断层扫描（ECT），完整基体的结构也已在原位以几纳米的分辨率被揭示。在这里，我们描述了MS环纯化、沙门氏菌微小细胞培养以及cryoEM/ECT数据收集和图像分析的方法。
