ArticleMethods in molecular biology (Clifton, N.J.)2022
Cryo-EM Analysis of the Lipopolysaccharide Flippase MsbA.
Article in Methods in molecular biology (Clifton, N.J.), 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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 LptB
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