In situ structural analysis of tripartite efflux assemblies by cryoET
1Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, TX, United States.
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The rise of antibiotic-resistant bacteria poses a critical threat to public health. A key mechanism by which bacteria acquire resistance is through multidrug efflux pumps that expel toxic compounds under antibiotic pressure. Among these, AcrAB-TolC (composed by AcrA, AcrB and TolC, with AcrB belongs to RND family) and MacAB-TolC (composed by MacA, MacB and TolC, with MacB belongs to ABC family) represent two major families of tripartite efflux pump systems in Gram-negative bacteria, each utilizing the same outer membrane channel TolC but differing in their inner membrane components and energization sources. Understanding assembling and functioning mechanism of these pumps requires cellular environment and precise conformational coordination for effective operation. Electron Cryo-tomography (cryoET), in combination with subtomogram averaging, is a unique approach enable direct visualizing macromolecular assemblies within native cellular contexts at subnanometer resolution without any purification, providing critical insights into their in situ architecture, assembly, and function. In this chapter, we present a detailed protocol for the in situ structural characterization of both AcrAB-TolC and MacAB-TolC efflux pumps in Escherichia coli. This unified workflow is broadly applicable to other efflux pumps on other bacterial strains and provides a starting point for studying antibiotic resistance mechanisms.
