1.1.1.282 | purified enzyme with bound NAD+ in a binary complex, and as ternary complexes with NADH plus either shikimate or quinate, sitting drop vapour diffusion method, for the ternary complexes: mixing of 0.002 ml of 10 mg/ml protein in 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20% v/v glycerol, with 1 mM NAD+/NADH or additonally with 35 mM of either quinate or shikimate, with 0.002 ml of crystallization solution containing for the QSDH-NAD+ crystals 1.6 M trisodium citrate, pH 6.5-6.9, 25-62 mM CoCl2, or for crystals of the QSDH-quinate-NADH and QSDH-shikimate-NADH 24% w/v polyethylene glycol 6000, 360-400 mM CaCl2, 100 mM Tris-HCl, pH 8.0-9.5. Crystals are soaked in a cryoprotectant containing 30% w/v polyethylene glycol 6000, 25% v/v glycerol, 100 mM Tris-HCl, pH 8.5, for 1 h, X-ray diffraction structure determination and analysis at 1.0-1.6 A resolution, structure modelling |