Cryo-EM Analysis of the Lipopolysaccharide Flippase MsbA.

MsbA is a member of the ATP-binding cassette (ABC) transporter family and harnesses the energy from adenosine triphosphate (ATP) binding and hydrolysis to flip lipopolysaccharide (LPS) across the cytoplasmic membrane in Gram-negative bacteria. MsbA is an essential component of the bacterial envelope biogenesis pathway and an attractive target for developing novel antibiotics against multidrug-resistant strains. Structural characterization of MsbA in different conformations provides crucial insights in understanding druggable pockets and mechanisms of inhibition of this transporter. Recent advances in membrane-mimetic environments and cryo-EM data acquisition and processing have enabled high-resolution imaging of MsbA in complex with its native LPS substrate. Despite these technical advances, MsbA remains a challenging target for cryo-EM analysis due to its small size and extraordinary conformational flexibility. Herein, we provide a protocol for the purification and incorporation of MsbA in lipid nanodiscs, cryo-EM sample preparation, and cryo-EM image processing. The method outlined here is generalizable to the study of other bacterial ABC transporters, including the LPS extractor LptB2FGC.