   1.14.11.56 | purified recombinant enzyme MlP4H in complex with Co2+, 2-oxoglutarate and L-Pro or L-Pip, the MlP4H protein used for crystallization includes an extra (Met)-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Gly-Lys-Gly-Ala strep-tag II peptide at its N-terminus, and the original start codon of the wild-type is replaced by the underlined alanine, followed by the second codon of the wild-type. Crystallization of L-Pro complex crystals by sitting-drop vapor diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM 2-oxoglutarate, and 20 mM L-Pro with reservoir solution containing 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium malonate, and 25% v/v PEG 3350, or of L-Pip complex crystals by sitting drop vapour diffusion method mixing 0.001 ml of 28 mg/ml protein solution containing 2 mM CoCl2, 10 mM ?2-oxoglutarate, and 20 mM L-Pip with 0.001 ml of reservoir solution containing 0.1 M CAPS, pH 10.5, 0.1 M lithium sulfate, and 1.8 M ammonium sulfate, all at 15°C, X-ray diffraction structure determination and analysis at 1.3-2.8 A resolution, by single-wavelength dispersion method with the bound Co2+ at the active site used as the anomalous scatter or by molecular replacement using the first L-Pro-bound structure as a search model |
743931 |
