   3.1.3.7 | purified recombinant Mycobacterium tuberculosis enzyme CysQ in a ligand-free structure, a lithium-inhibited state with substrate PAP bound, and a product-bound complex with AMP, phosphate, and three Mg2+ ions bound, sitting drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT, 5% glycerol, and 1 mM AMP, with 0.001 ml of reservoir solution containing 24% PEG 1500 and 20% glycerol for the ligand-free enzyme, the calcium- and phosphate-bound structure is grown in 0.05 M CaCl2, 0.1 M Bis-Tris-HCl, pH 6.5, and 30% PEG MME 550, the magnesium- and phosphate-bound structure is grown in 0.1 M Mg acetate, 0.1 M Bis-Tris-HCl, pH 6.5, and 35% PEG MME 550, and for both the Ca2+ and Mg2+ structures, phosphate is not included in the crystallization conditions, the AMP-, Pi-, and Mg2+-bound crystals are grown in 0.1 M Mg acetate, 0.1 M Bis-Tris-HCl, pH 6.5, and 35% PEG MME 550 and soaked for 20 min in mother liquor supplemented with 3 mM AMP, 21°C, 1-5 days, X-ray diffraction structure determination and analysis at 1.45-2.05 A resolution. SIRAS (single isomorphous replacement with anomalous scattering) phasing information is collected from a ligand-free crystal grown in 0.2 M calcium acetate, 0.1 M MES-NaOH, pH 6.0, and 20% w/v PEG 8000 that is soaked overnight in mother liquor supplemented with a heavy atom cocktail: 1 mM uranyl acetate, 1 mM p-chloromercury benzoic acid sulfonate (PCMBS), and 1 mM KAu(CN)2. Before being flash-cooled, the crystal is transferred to a cryosolution containing 0.2 M Ca-acetate, 0.1 M MES-NaOH, pH 6.0, 20% w/v PEG 8000, and 20% glycerol. Molecular replacement and structure modelling |
