Crystallization of Silver-Loaded CusS Protein

The pTXB3-CusS_(39-187) plasmid was used for expression of CusS_(39-187)^{11 (link)}. Apo CusS_(39-187) and silver loaded CusS_(39-187) (Ag(I)-CusS_(39-187)) were prepared as previously described^{11 (link)}. Ag(I)-CusS_(39-187) in 25 mM MES pH 6.0 buffer was concentrated to 7–8 mg/mL for crystallization. Initial crystallization of Ag(I)-CusS_(39-187) was performed with commercially-available Index HT screening solutions (Hampton Research). Droplets consisting of 0.2 μL protein solution and 0.2 μL of each precipitant solution were mixed in sitting drops in a 96-well plate using a PHOENIX protein crystallization robot (Art Robbins Instruments). Clusters of rod-shaped crystals of CusS_(39-187) formed after one week in a precipitant solution containing 100 mM Tris-HCl pH 8.5, 200 mM ammonium acetate, and 25% PEG-3350 at 4°C. The crystallization conditions were further optimized in the initial precipitant solution with varying PEG-3350 percentages and protein:precipitant ratios using sitting-drop vapor-diffusion at 4°C. Clusters of crystals were observed in all drops containing 100 mM Tris-HCl pH 8.5, 200 mM ammonium acetate, and 25% PEG-3350 with 1:2 protein:precipitant ratio. Diffraction quality protein crystals were obtained by equilibrating droplets against 1 mL precipitant solution using the sitting-drop vapor-diffusion method at 4°C. Crystals were flash frozen in liquid nitrogen.