Crystallization

Recombinant Pvg1p was purified as described previously²⁵ with the following modifications. Ni-affinity and gel filtration chromatographic procedures were performed using a HisTrapTM FF (1 mL) column and a Superose 6 10/300 GL column (GE Healthcare), respectively. Single crystals were grown by the hanging-drop vapor diffusion method at 20 °C. The protein solution was composed of 10 mg/mL Pvg1p in 100 mM MOPS-NaOH (pH 7.4) containing 20 mM PEP and 5 mM pNP-β-Gal. The well solution contained 200 mM magnesium choloride, 10 mM zinc chloride, 26% (w/v) Poly(acrylic acid sodium salt) 5100 in 0.1 M HEPES (pH 7.5) buffer. Each hanging-drop consisted of 1 μL of protein solution and 1 μL of well solution.