In Situ Structural Analysis of the Spirochetal Flagellar Motor by Cryo-Electron Tomography

Abstract

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.

Keywords: Cryo-electron tomography; Flagellar motor; Periplasmic flagellum; Spirochete; Sub-tomogram averaging.