   1.1.3.12 | purified recombinant C-terminally His6-tagged PNOX and PNOX-pyridoxamine complex, sitting drop vapour diffusion mthod and micro-seeding method, mixing of 0.002 ml of 10 mg/ml PNOX with 0.002 ml of a precipitant solution comtaining 0.1 M HEPES-NaOH, pH 7.5, 10% v/v 2-propanol, and 20% w/v PEG 4000, equilibration against 01 ml of precipitant solution, 4°C, primary crystals are crushed and suspended in 0.5 ml of a solution consisting of equal volumes of a crystallization buffer containing 50 mM Tris-HCl, pH 8.0, 10% w/v glycerol, 0.005 mM FAD, 0.1% v/v 2-mercaptoethanol, and 0.01% v/v Tween 20, and precipitant solution as seeding solution, or seeding crystals are prepared by vapor diffusion in a mixture consisting of 0.002 ml of 13 mg/ml PNOX, and 0.002 ml of the precipitant solution comprising 0.1 M HEPES-NaOH, pH 7.5, 25% v/v 2-propanol, 20% w/v PEG 4000, and 10 mM pyridoxamine, 3 weeks, X-ray diffraction structuure determination and analysis at 2.2 A and 2.1 A resolutions, respectively, molecular replacement |
