Sg1 screen

Before crystallization, OXA-436 (13.8 mg ml⁻¹) was dialyzed into a buffer consisting of 50 mM HEPES pH 7.2. A single crystal was observed from condition F2 (25% PEG 3350, 0.2 M sodium acetate, 50 mM HEPES pH 7.5) of the sparse-matrix SG1 screen (Molecular Dimensions) after incubation at room temperature for three weeks. A plate-like crystal (Fig. 1 ▸) was harvested from a grid screen optimizing the PEG 3350 concentration and the pH. A cryosolution was prepared consisting of 0.1 M HEPES pH 8, 25% PEG 3350, 0.2 M sodium acetate, 25% ethylene glycol, and the crystal was flash-cooled in liquid nitrogen. Additional crystallization information is summarized in Supplementary Table S2.

Initial crystallization screening was performed using the sitting-drop vapor-diffusion method with a Mosquito crystallization robot (TTP Labtech, Melbourn, UK) and several commercially available screening kits such as MCSG I-IV (Microlytic, Burlington, MA, USA), SaltRx, Index (Hampton Research, Aliso Viejo, CA, USA), and SG1 Screen (Molecular Dimensions, Suffolk, UK). Each crystallization drop consisted of 200 nL protein solution and 200 nL reservoir solution against 80 µL reservoir solution in 96-well sitting-drop plates (Emerald Bio, Bainbridge Island, WA, USA) at 293 K. CYP153D17 was crystallized under several different conditions within two days. To obtain optimal crystals, the crystallization condition of 25% (w/v) PEG 3350 (SG-1 #E2) was varied according to the precipitant concentration by using the hanging-drop vapor-diffusion method in 24-well crystallization plates (Molecular Dimensions) at 293 K. The drop volume was increased to 1 µL protein and 1 µL reservoir solution against 500 µL reservoir solution. An optimal single crystal was obtained using 22% (w/v) PEG 3350 and had a rectangular stick shape. The single crystal was picked using a loop and mounted without cryoprotectant. Diffraction data were collected using a synchrotron-radiation source at the BL-7A beam line, Pohang Accelerator Laboratory, Pohang, Korea.