Structural Study of the Bacterial Flagellar Basal Body by Electron Cryomicroscopy and Image Analysis

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.