Crystal screening kits

Crystallization of the apo EcYjgB was initially performed using crystal screening kits (Hampton Research Co., Aliso Viejo, CA, USA and Emerald Biostructures Co., Bainbridge Island, WA, USA) and by using the sitting-drop vapor-diffusion method at 20 °C. Each experiment consisted of mixing 1.0 μL of the protein solution with 1.0 μL of reservoir solution and equilibrating this against 0.1 mL of the reservoir solution. Thin plate-shaped crystals were observed after 1–3 days. The diffraction of these crystals was not suitable for the determination of their structure. We adjusted the concentration of the precipitant, polyethylene glycol (PEG) 3350, and the pH of the reservoir solution to obtain high purity for crystallization. The suitable crystals for diffraction were obtained under conditions of 0.1 M Tris-HCl, pH 7.0, 16% (w/v) PEG 3350, and 0.2 M lithium sulfate. The suitable crystals grew within 1–3 days to dimensions of approximately 200 × 50 × 10 μm. The apo EcYjgB crystals were soaked into the crystallizing buffer supplemented with 5 mM NADP for 30 min. at 7 °C to enable the formation of the EcYjgB-NADP complex. In this case, apo-form crystals were prepared within 1 day by hanging-drop vapor diffusion method at 20 °C, under the condition of 0.08 M Ammonium citrate tribasic pH 7.0 and 18% PEG 3350.