   2.7.1.232 | purified recombinant enzyme, hanging drop vapor diffusion method, dimeric enzyme: mixing of equal volumes of 7 mg/ml protein in 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5mM TCEP, and 20mM Tris, pH 7.5, with reservoir solution containing 18% PEG 4000, 100 mM sodium acetate, 100 mM Tris, pH 7.2, for the free enzyme and with reservoir solution containing 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5 mM TCEP, 1 mM aluminum nitrate, 10 mM sodium fluoride, 20 mM Tris, pH 7.5, for the complexed enzyme. Monomeric enzyme: 9 mg/ml protein in 50 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, and 200 mM levoglucosan is mixed with reservoir buffer containing 1.8 M ammonium sulfate, HEPES, pH 7.0, and 100 mM sodium acetate for th free enzyme, and 25 mg/ml protein in 100 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, 2 mM ADP, 4 mM MgCl2, 1 mM aluminum nitrate, and 10 mM sodium fluoride, with reservoir buffer containing 1.3 M sodium malonate, 93 mM Bis-Tris propane, pH 7.0, for the complexed enzyme, soaking of resultant crystals in the drop with 200 mM levoglucosan. X-ray diffraction structure determination and analysis at 1.5-2.0 A resolution |
