    4.1.99.11 | purified recombinant trimeric enzyme BSS in complex with fumarate and toluene or recombinant dimeric BSSalphagamma enzyme form in complex with fumarate, sitting drop vapor diffusion method, mixing of 8 mg/ml trimeric enzyme in 50 mM Tris, pH 7.6, 15% v/v glycerol, and 200 mM NaCl, in a 2:1 ratio with well solution containing 25% w/v PEG 3350, 100 mM Tris, pH 8.5, 60 mM KCl, and 5 mM fumarate, 0.001-0.002 ml of toluene is added to the bottom of the well and allowed to diffuse slowly into the protein drop, 3 weeks at room temperature. Mixing of 15 mg/ml dimeric enzyme in 20 mM HEPES, pH 7.6, 100 mM NaCl, and 5 mM fumarate, in a 1:1 ratio with well solution containing 20% w/v PEG 400, 50 mM Bis-Tris, pH 6.5, and 25 mM Tris, pH 8.0, several days at room temperature, soaking of crystals in mother liquor with ligands at 50 mM, X-ray diffraction structure determination and analysis at 3.3 A and 2.0 A resolution, respectively, modelling |
