MexB/inhibitor complexes preparation for crystallography and cryo-EM, comparison and possible pitfalls.

In the context of the increasing appearance of bacterial antibiotic resistance, there is an urgent need to discover new molecules that target bacterial defenses to antibiotics. One of the scientific strategies for developing new molecules is the use of structural biology for drug design. In this chapter, we focus on the study of one of the bacterial defenses, the RND efflux pumps, able to expel antibiotics and other unwanted molecules out of the cell before those noxious compounds reach their target. We will present the protocols we used to solve the structures of complexes between synthesized chemicals and MexB, the inner membrane transporter involved in the formation of the RND efflux pump MexAB-OprM from Pseudomonas aeruginosa. The structural analyses were performed both by crystallography and by cryo-electron microscopy, and we highlight the strategic parameters inherent to the two methods that need to be taken into account for successful structure determination of the complexes.