3.4. Crystallization and Structure Determination

Crystallization experiments were performed using the sitting-drop vapor-diffusion method at 14 °C. Before crystallization, the MerTK kinase domain protein stock was incubated with 5 mM AZD7762 (approximately 1:5 protein:compound molar ratio) at 4 °C overnight. Crystallization drops were set up by mixing 0.5 µL of the compound–protein solution and 0.5 µL of reservoir solution (100 mM Tris-HCl, pH 8.5, 4 M sodium chloride). Crystals grew within 5 days.

Diffraction data were collected using a Dectris Pilatus 6M CCD detector at the BL-11C experiment station of the Pohang Light Source (Pohang, Korea). Crystals were cryoprotected using cryobuffer (reservoir solution supplemented with 2.5 mM AZD7762 and 30% (v/v) glycerol). Diffraction data were processed and scaled using the HKL2000 program suite [40]. The structure was solved by molecular replacement with PHASER [41] using a MerTK structure (PBD entry, 4MH7) as the search model [42]. Model building was completed using COOT [43], and refinement was performed with phenix.refine in the Phenix program suite [44]. Coordinates and cif restraint files for AZD7762 were generated using Maestro version 2017-3 (Schrödinger, New York, NY, USA) and eLBOW in the Phenix program suite, respectively [45,46]. Data collection and refinement statistics are listed in Table 1. The structural figures were drawn using Pymol, ver. 2.3.0 (Schrödinger, New York, NY, USA).