Crystallization

Purified SsDesB (12 mg/mL in 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 25 mM imidazole) was screened for crystals using several sparse-matrix crystallization screens from Hampton Research, Microlytic, Qiagen, and Molecular Dimensions using a Gryphon crystallization robot (Art Robbins Instruments; Sunnyvale, CA). Optimization screens of the initial crystallization screening hits were carried out using the hanging-drop vapor diffusion method in Easy-Xtal 15-well plates (Qiagen; Germantown, MD). Additional optimization screens were completed using the Hampton Research Additive screen. Crystals of SsDesB for data collection were obtained by mixing 2.5 μL of protein solution (12.1 mg/mL) with 2 μl well solution (0.1 M Hepes pH 7.5, 0.2 M sodium chloride, 25% w/v polyethylene glycol 3350), and 0.5 μL of 0.1 M taurine and sealing over 500 μL of well solution. The trays were incubated at 4°C. Yellow, plate-like crystals appeared within 1 week. A single crystal was retrieved from a drop using a Litholoop (Molecular Dimensions; Maumee, OH) and transferred to a 1 μl drop of Paratone-N where excess mother liquor was whisked away from the crystal. The crystal was removed with a Litholoop and flash-cooled by plunging it into liquid N₂.

Crystals of SsDesB in complex with NADP⁺ were obtained by incubating 12.1 mg/mL of protein with 1 mM NADP⁺ (Sigma-Aldrich, St. Louis, MO) for 2 hrs at 4°C. Precipitated material was removed by centrifugation for 10 min and 2.5 μL of the SsDesB:NADP⁺ mixture was mixed with 2.0 μL of well solution (0.1 M Hepes pH 7.5, 0.2 M sodium chloride, 25% w/v polyethylene glycol 3350) and 0.5 μL (0.1 M sarcosine) and sealed over 500 μL of well solution. After 1 hr, the drops were streak-seeded with a whisker using previous crystals of SsDesB as a seed source. Yellow, plate-like crystals appeared within 2 days. A single crystal for data collection was retrieved using a Litholoop, transferred to a 1 μL drop of Paratone-N, where excess mother-liquor was whisked away from the crystal, and the crystal was flash-cooled by plunging into liquid N₂.