Cryo-EM structural analysis of liposome-reconstituted AcrB in the presence of substrates.

AcrB is a member of the resistance-nodulation-cell division (RND) family of efflux transporters expressed in Escherichia coli, and it plays a crucial role in antimicrobial resistance. AcrB has been structurally characterized in a variety of states through observations using diverse methods, and by applying conditions that have not yet been explored, there is potential to reveal new structural features. In this study, we performed cryo-electron microscopy (cryo-EM) structural analysis of AcrB reconstituted into liposomes in the presence of the substrate doxorubicin (DOX). As a result, both the functional LTO state and the resting LLL state were observed on the same EM grid. In the LTO state, substrate binding was observed in the deep binding pocket of the T protomer, whereas the LLL state could be further classified into multiple subclasses. This study not only provides additional structural insight into AcrB but also highlights the importance of observing membrane proteins under a variety of conditions.