   2.7.1.151 | purified recombinant core catalytic domain of HsIMPK, that contains residues 50 to 416, from which an internal domain comprising residues 263 to 377 is deleted and replaced with a simple Gly-Gly-Ser-Gly-Gly linker, apostructure and catalytic core domain with bound ADP-Ins (1,4,5)P3-Mg and ADP-DiC4-PtdInsP2-Mg, hanging drop vapor diffusion, mixing of 0.002 ml of 38 mg/ml protein solution with 0.002 ml of well solution containing 35% w/v PEG 400, 0.1 M Li2SO4, 100 mM MES-imidazole, pH 6.0, and 50 mM 2-mercaptoethanol, 25°C, to obtain complex structures, apoenzyme crystals are further soaked for 1 day in 35% w/v PEG 400, 100 mM Li2SO4, 100 mM HEPES, pH 7.5, at 25°C, in the presence of 20 mM Ins(1,4,5)P3 or a soluble diC4-analogue of PtdIns(4,5)P2, 10 mM MgCl2, and 5 mM of either Na2ATP or Li2AMP-PNP, X-ray diffraction structure determination and analysis at 1.63-1.93 A resolution. The structure of the IPMK apoenzyme is determined by a molecular replacement approach using a model constructed from the template of yeast ScIPMK (PDB ID 2IF8) |
