Crystallization of RoqN.

Crystals of both native and SeMet RoqN were grown at 20 °C by sitting drop vapor diffusion in Intelli-Plate™ 96–2 shallow well plates (Hampton Research) by combining 1 μL RoqN at 12 mg/mL and 1 mM SAM in storage buffer with 1 μL of well solution composed of 19% PEG 3350, 2% ethylene glycol, 200 mM Mg(C₂H₃O₂)₂. Droplets were immediately nucleated using a cat whisker charged with RoqN crystals from an earlier crystallization event. The crystals were cryoprotected by the direct addition of 10 μL of a cryoprotectant solution composed of 20% PEG 3350, 23% ethylene glycol, 200 mM Mg(C₂H₃O₂)₂, 50 mM NaCl, 10 mM HEPES pH 7.6, 1 mM SAM to the crystallization droplet. Crystals were flash frozen in liquid nitrogen. RoqN was solved by single anomalous dispersion (SAD) in the CCP4 software package³⁹ by using the Crank2 pipeline⁴⁰ in the default configuration. The initial model was subjected to iterative rounds of building and refinement using Coot³⁶ and Refmac ³⁷.