Protein crystallization

Crystals of MmMK have been grown previously [18], and a resulting apo-MmMK structure was deposited in the Protein Data Bank (PDB entry 4HAC). However, because we were not able to reproduce crystals from these published conditions, we identified new crystallization conditions for mevalonate-bound MmMK via sparse-matrix screening by sitting-drop vapor diffusion using a Crystal Gryphon (Art Robbins Instruments) with 11 mg/mL MmMK in storage buffer. Crystals were observed in 100 mM Bis-Tris pH 5.5, 200 mM sodium potassium tartrate, and 30% polyethylene glycol (PEG) 3,350. Crystallization conditions then were optimized by hanging-drop vapor diffusion with varying sodium potassium tartrate, PEG molecular weight, and PEG concentrations. The best crystals grew overnight using 15–25% PEG 8,000 or 10,000. Crystals were then looped, washed in mother liquor, crushed via vortexing, and used as seeds for microseeding. Seeded drops contained 1.0 μL of the MmMK sample with 1 mM (R)-mevalonate, 5 mM AMPPNP, and 5 mM MgCl₂, 0.8 μL crystallization solution, and 0.2 μL of the seed stock. Diffraction-quality plate crystals grew in crystallization solutions that contained 100 mM Bis-Tris pH 5.5, 200–300 mM sodium potassium tartrate, and 15–25% PEG 8,000 or 10,000. Crystals were cryoprotected using 100 mM Bis-Tris pH 5.5, 20% PEG 8,000 or 10,000, 20% glycerol, 1 mM (R)-mevalonate, 5 mM AMPPNP, and 5 mM MgCl₂ before flash-cooling in liquid nitrogen. Despite the presence of AMPPNP at 5 mM concentrations, the resulting structures did not yield any electron density in the ATP binding site, similar to prior crystallographic studies on LmMK [12].

Crystals of MmMK with (R)-5-phosphomevalonate were grown and optimized using the same crystallization solution as with (R)-mevalonate-bound MmMK, with 1 mM (R)-5-phosphomevalonate and 5 mM MgCl₂.