Crystallization of D417C Mutant Protein

For crystallization, the D417C mutant was put into a deletion construct (ΔNC) lacking N-terminal residues 2–16 and C-terminal residues 461–464 (Lim et al., 2012 (link)). Purified ΔNC-D417C was cross-linked with 100 µM CuP for 1 h, incubated with excess Fab fragment (Dutzler et al., 2003 (link)) for 30 min, then purified by size exclusion chromatography (Superdex 200) into buffer containing 100 mM NaCl, 5 mM DM, 10 mM Tris (Fisher Scientific, Pittsburgh, PA), pH 7.5. The complex was concentrated to 10–12 mg/mL and mixed with 30% PEG 400 (Hampton Research, Aliso Viejo, CA), 0.075 M K/Na-tartrate (Fluka Analytical, Ronkonkoma, NY), 0.1 M Tris HCl (MP Biomedicals, Santa Ana, CA) (pH 9.0). Crystals were grown by the sitting drop method for 2–4 weeks at 20^oC and were directly harvested from the reservoir, flash frozen and stored in liquid N₂. Diffraction data were collected to 0.9795 Å at the BL12-2 beamline (SLAC) and processed using XDS (Kabsch, 2010 (link)). Phases were obtained by molecular replacement with the WT protein in complex with Fab (PDB 1OTS) using the MOLREP program (Vagin and Teplyakov, 2010 (link)). Refinement was done using the refmac program (Murshudov et al., 1997 (link)). Atomic coordinate and structure factors are deposited in the Protein Data Bank under accession code 5HD8.