   1.13.11.66 | purified enzyme free or in complex with substrate methylhydroquinone under anaerobic conditions, or with inhibitors 4-hydroxybenzoate and 4-nitrophenol, sitting drop vapor diffusion method, mixing of 0.001 ml of 8 mg/ml protein in 25 mM Tris, pH 7.0, 5 mM NaCl, and 0.5 mM ligand, with 0.001 ml of reservoir solution containing 14% PEG 3350, 0.35 M MgCl2, and 0.1 M MES, pH 6.5, 4°C, 1 day, the structure of the free enzyme is obtained by soaking the crystals in a stabilizing solution containing 16% PEG 3350, 0.35 M MgCl2, and 0.1 M MES, pH 6.5, for two days, changing the solution three times, in order to remove the ligands, X-ray diffraction structure determination and analysis at 1.90-2.40 resolution, molecular replacement using the coordinates of PnpCD structure from Pseudomonas sp. strain WBC-3, PDB ID 4ZXA as template, modeling |
| 1.13.11.66 | purified full-length or proteolytically truncated PnpCD in apo form and in complex with Fe3+ or substrate analogue hydroxybenzonitrile and Cd2+, i.e. apo-PnpCD, PnpCD-Fe3+, and PnpCD-Cd2+-HBN, sitting-drop vapor diffusion method, 15-20 mg/ml protein in 10mM Tris-HCl, pH 8.0, and 100 mM NaCl, is mixed with reservoir solution containing 0.2 M sodium thiocyanate, 20% w/v PEG 3350, 20°C, method optimization, X-ray diffraction structure determination and analysis |
