EC Number   |
|---|
    2.7.1.50 | crystallization by vapor diffusion equilibration, crystal structure at 1.5 A resolution |
| 2.7.1.50 | enzyme with a phosphate ion occupying the position of the beta-phosphate of the nucleotide, mixing of 0.001 ml of protein solution containing 1.95 mg/ml protein in 20 mM Tris-HCl containing 200 mM NaCl and 1 mM DTT, pH 8.0, with 0.001 ml of reservoir solution containing 0.1 M CHESS buffer, pH 9.3, and 0.88 M sodium citrate as the precipitant, 20°C, 1 week, 25% v/v glycerol for cryoprotection, X-ray diffraction structure determination and analysis at 1.85 A resolution |
| 2.7.1.50 | precipitant: sodium citrate, pH 9.3, 293 K |
| 2.7.1.50 | purified enzyme SaThiM in apoform and in complexes with the natural substrate 5-(hydroxyethyl)-4-methylthiazole (THZ) and two selected substrate analogues, cpd1 and cpd2, sitting drop vapor diffusion method using 10-20 mg/ml protein in 100 mM Tris, and 150 mM NaCl, pH 8.0, with an equal volume of 0.5 M magnesium formate, microcrystal seeding, with precipitant solution containing 18, 20 or 22% PEG 3350 w/v, 0.2 M magnesium formate, and 5% isopropanol (v/v), against a 0.5 ml reservoir of precipitant, X-ray diffraction structure determination and analysis at 1.62-2.09 A resolution. The homologue structure of Bacillus subtilis ThiK (PDB ID 1C3Q), sharing 38% sequence identity, is used as a search model for molecular replacement phasing |
| 2.7.1.50 | purified recombinant detagged enzyme, in complex with beta,gamma-methyleneadenosine 5'-diphosphate, thiamin phosphate, 4-amino-5-hydroxymethyl-2-trifluoromethylpyrimidine diphosphate, or 4-methyl-5-hydroxyethylthiazole phosphate, hanging-drop vapor diffusion method, mixing of0.001 ml protein solution with 0.001 ml of reservoir solution containing 0.1 M HEPES, pH 7.5, 0.2-0.25 M MgCl2, and 25-32% PEG400, 22°C, 2-3 days, method optimization, X-ray diffraction structure determination and analysis at 2.6-3.3 A resolution, modeling |
