Overexpression of the Multidrug Efflux Pump AcrAB-TolC in Escherichia coli for In Situ Cryo-ET Study

The emergence of antibiotic-resistant bacteria has become a crucial clinical challenge. Bacteria acquire resistance by employing multidrug efflux pumps to expel toxic substances and survive under antibiotic pressure. Among the efflux pumps, AcrAB-TolC represents a major type of resistance-nodulation-cell division efflux pump existing in Gram-negative bacteria, which requires an intact cellular environment and intricate conformational changes to effectively expel antibiotics. To date, cryo-electron tomography and subtomogram averaging stand as a unique structural biology method capable of achieving nanometer resolution 3D structural details in native cells. These advantages make it the preferred approach for investigating complex in situ assemblies and conformations within physiological cellular contexts. Here, we outline a detailed protocol for studying the in situ structure of the AcrAB-TolC efflux pump in Escherichia coli. The protocol applies to other tripartite systems in various bacterial strains, providing a comprehensive pipeline for in situ structural studies and enhancing the development of potential therapeutics.