   2.7.1.170 | purified recombinant enzyme bound to a nonhydrolyzable ATP analogue (AMPPCP) and 1,6-anhydroMurNAc, hanging drop vapor diffusion method, for enzyme AnmK bound to AMPPCP, 6.5 mg/ml protein in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, and 20 mM Tris, pH 8.0, is mixed with an equal volume of mother liquor containing 26% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 9.0, for enzyme AnmK bound to AMPPCP and the anhMurNAc sugar, AnmK s first co-crystallized with AMPPCP by mixing equal volumes of mother liquor containing 24% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 8.2, with the enzyme at 6.5 mg/ml in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, 20 mM Tris, pH 8.0, a single crystal of the AnmK-AMPPCP complex is then transferred to a drop of reservoir buffer supplemented with 8 mM anhMurNAc, 2 mM AMPPCP, and 4 mM MgCl2 and soaked for 1 h, at room temperature, X-ray diffraction structure determination and analysis at 1.67-1.84 A resolution |
