The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porintype open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-Å resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring ␤-barrel and the cavity-forming ␣-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH 2 -terminal fatty acids and the other is the ␤-barrel structure consensus on the outer membrane-spanning proteins. The ␤-barrel had a pore opening with a diameter of about 6 -8 Å, which is not large enough to accommodate the exit of any antibiotics. The periplasmic ␣-barrel was about 100 Å long formed mainly by a bundle of ␣-helices that formed a solvent-filled cavity of about 25,000 Å 3 . The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.

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Crystals of the drug-discharge outer membrane protein OprM (MW = 50.9 kDa) of the MexAB-OprM multidrug transporter of Pseudomonas aeruginosa have been grown at 293 K in the presence of 2-methyl-2,4-propanediol and a combination of surfactants. The crystal belonged to space group R32, with unitcell parameters a = b = 85.43, c = 1044.3 A Ê. Diffraction data for OprM were obtained using the undulator synchrotron-radiation beamline at SPring-8 (BL44XU, Osaka University), which allowed an extra-long specimen-todetector distance with a wide detector area. The crystal diffracted to 2.56 A Ê resolution using 0.9 A Ê X-rays from the synchrotron-radiation source. A heavyatom derivative for isomorphous replacement phasing was obtained using iridium chloride. crystallization communications Acta Cryst. (2005). F61, 131±133 Akama et al. OprM 133 Figure 2 Difference Patterson map in the Harker plane. (a) shows the isomorphous difference map and (b) the Bijvoet anomalous difference map. Both peaks are from atomic vector site A (x, y, z) = (0.0645, 0.4028, 0.3220). The maps were contoured at intervals of 1.0 times the r.m.s. density of the map.