In Pseudomonas aeruginosa , MexAB–OprM plays a central role in multidrug resistance by ejecting various drug compounds, which is one of the causes of serious nosocomial infections. Although the structures of the components of MexAB–OprM have been solved individually by X-ray crystallography, no structural information for fully assembled pumps from P. aeruginosa were previously available. In this study, we present the structure of wild-type MexAB–OprM in the presence or absence of drugs at near-atomic resolution. The structure reveals that OprM does not interact with MexB directly, and that it opens its periplasmic gate by forming a complex. Furthermore, we confirm the residues essential for complex formation and observed a movement of the drug entrance gate. Based on these results, we propose mechanisms for complex formation and drug efflux.

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α‐glucosidase. Here, we determined the crystal structure of Flavobacterium johnsoniae dextranase (FjDex31A), an enzyme from F. johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N‐terminal domain, a catalytic domain, a proximal C‐terminal domain, and a distal C‐terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite −1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α‐glucose residue at subsite −2, as well as Trp376 and Leu308‐cisGln309, are especially unique to FjDex31A. Site‐directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite −2 is likely to be important for its activity. Database Structural data are available in the Protein Data Bank under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6JR6, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6JR7, and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6JR8.

Graphical Abstract Highlights d Viruses in the family Reoviridae possess a symmetry mismatched multilayer capsid d An assembly intermediate was prepared using assembly defect outer capsid proteins d Structure was examined using conventional and Zernike/ Volta phase-contrast cryo-EM d Structure of the stalled intermediate revealed the mechanism of second-layer assembly