Purification and Sonication-Assisted Cryo-EM Grid Preparation of TolC.

TolC is a multifunctional outer membrane protein essential for bacterial physiology. As the outer membrane component of tripartite efflux complexes, TolC mediates antibiotic resistance while also serving critical roles in protein secretion and metabolite transport. Understanding these diverse functions through structural and functional studies is critical for developing new antimicrobial therapies and overcoming drug resistance. Structural analysis of TolC has been predominantly conducted using X-ray crystallography. However, since TolC functions through complex formation with numerous periplasmic and secreted proteins in cells, cryogenic electron microscopy (cryo-EM) is more suitable for analyzing these transient complexes. This chapter describes purification strategies for TolC and its protein complexes, as well as cryo-EM sample preparation protocols, including sonication treatment to improve particle distribution. We also address challenges specific to membrane protein structural biology and discuss structural analysis and validation methods with detailed methodological updates.