   6.3.2.36 | purified free enzyme, and enzyme in complexes with ATP, and ATP and 4-phosphopantoate, sitting drop vapor diffusion method, for the free enzyme: mixing of 0.001 ml of 20 mg/ml of protein in 50 mM Tris-HC, pH 8.0, and 150 mM NaCl, with 0.001 ml of reservoir solution containing 18-20% PEG 3350, 200 mM ammonium acetate, and 100 mM Bis-Tris, pH 6.5, 20°C, 1 week, method optimization, for the enzyme-ATP-Mg2+ complex: 20 mg/ml protein is incubated for several hours at 4°C in the presence of 10 mM ATP and 10 mM MgCl2, mixing of 0.001 ml protein solution with 0.001 ml of reservoir solution containing 17% PEG 10000, 100 mM ammonium acetate, and 100 mM Bis-Tris, pH 5.5, equilibration with reservoir solution, 20°C, 2 weeks, for the enzyme-ATP-Mg2+-4-phosphopantoate complex, mixing of 0.001 ml of 20 mg/ml of protein in 50 mM Tris-HC, pH 8.0, 150 mM NaCl, mM ATP, 5 mM MgCl2, and 5 mM PPo, with 0.001 ml of reservoir solution containing 50% tacsimate, pH 4.0, 1% PEG 3350, and 100 mM sodium acetate trihydrate, pH 4.5, 4°C, 3 days, X-ray diffraction structure determination and analysis at 2.0-2.4 A resolution |
