    6.3.1.21 | purified enzyme complexed with the nonhydrolyzable ATP analogue AMP-PNP and complexed with AMP-PNP and glycinamide ribonucleotide, hanging drop vapor diffusion method, mixing of 20 mg/ml protein in 15 mM HEPES, pH 7.5, 5 mM AMP-PNP, 10 mM MgCl2, and 250 mM NaCl, in an 1:1 ratio with reservoir solution containing 18-22% w/v methyl ether poly(ethylene glycol) 5000, 100 mM MOPS, pH 6.7, and 100 mM MgCl2, 4°C, 2-3 weeks, X-ray diffraction structure determination analysis at 1.9 A resolution. For preparation of the complex with GAR, crystals grown in the presence of AMP-PNP are transferred to a solution containing 20% w/v methyl ether poly(ethylene glycol) 5000, 50 mM MgCl2, 350 mM NaCl, 2.5 mM AMP-PNP, 100 mM MOPS (pH 6.7), and 1 mM GAR, the crystals are allowed to soak for 12 h, X-ray diffraction structure determination analysis at 1.75 A resolution. Structure modelling, detailed overview |
