Cl centrifugal filter unit

Initial crystallization conditions were identified in the polyethylene glycol (PEG)/ion HT Screen using a Phoenix pipetting robot (Art Robbins Instruments). Rectangular crystals formed within 24 to 48 h at 18 °C. Optimal crystals were obtained by sitting drop vapor diffusion in reservoir conditions of 12% to 18% (w/v) PEG 3350, and either 0.14 to 0.20 M succinate or 0.2 to 0.3 M sodium acetate set up at room temperature. Protein samples were prepared by spin filtration using a 0.22-μm Millipore CL Centrifugal Filter Unit. Drops at a ratio of 1 μl MsuD (8 mg/ml) to 1 μl reservoir were equilibrated against 600 μl of reservoir. Crystals ranging in size from 50 to 200 μm in the longest dimension were cryoprotected with increasing increments of glycerol to a final concentration of 15% to 20% (v/v) with an increase of 3% to 5% (w/v) PEG 3350. Crystals were mounted in loops and cryocooled in liquid nitrogen.

Structures of the ternary complex with FMN and MS⁻ (MsuD•FMN•MS⁻) were obtained by both soaking (ternary-soak MsuD) and cocrystallization (ternary-MsuD) experiments, yielding ternary complex structures in monoclinic and hexagonal space groups, respectively. For cocrystallization, MsuD at 8 mg/ml was incubated with 2 mM FMN and 2 mM MS⁻ on ice for 15 min prior to spin filtration using a 0.22-μm Millipore CL Centrifugal Filter Unit. Crystallization was performed in a sitting drop vapor diffusion experiment with 1 μl MsuD•FMN•MS⁻ mixture and 1 μl of reservoir (0.24 M succinate and 12% (w/v) PEG 3350). Hexagonal crystals ranging in size from 50 to 200 μm in the longest dimension formed within 24 to 48 h at 18 °C. For soaking experiments, MsuD crystals were soaked in reservoir solution supplemented with FMN and MS⁻. A volume of 2 μl reservoir solution supplemented with 2 mM FMN and 2 mM MS⁻ was added to a 1-μl drop containing MsuD crystals and incubated for over 16 h at 18 °C. Crystals were cryoprotected in the same manner as binary-MsuD, but with cryoprotectant supplemented with 2 mM FMN and 2 mM MS⁻, and cryocooled in liquid nitrogen.