   1.1.1.286 | purified recombinant His6-tagged enzyme in apoform or in complex with substrates isocitrate, homoisocitrate, and 3-isopropylmalate, hanging drop vapour diffusion method, for the apoenzyme: mixing of 0.001 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl, with 0.001 ml of reservoir solution containing 100 mM imidazole-HCl, pH 6.5, and 2.3 M NaCl, equilibration against 1 m of reservoir solution, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM homoisocitrate, with 0.0015 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 10% v/v 2-propanol, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 5 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM isocitrate, with 0.0015 ml of reservoir solution containing 100 mM TrisHCl, pH 7.5, 200 mM NaCl, and 30% v/v 2-methyl-2,4-pentanediol, at 20°C, and for the ternary complex with 3-isopropylmalate and Mn2+: mixing of 0.001 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM 3-isopropylmalate, with 0.001 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 36% v/v 2-methyl-2,4-pentanediol, at 20°C, X-ray diffraction structure determination and analysis at 1.7 A resolution for the apoenzyme, and at 2.50-2.64 A resolution for the complexed enzyme |
