A synthetic cDNA for human CYP17A1 was modified to delete residues 2–19, substitute the hydrophilic sequence 20RRCP23 (link) with 20AKKT23 (link), and add a C-terminal four histidine tag (fig. S6) before cloning into the pCWori+ plasmid and overexpression in E. coli JM109 cells. Protein was purified by nickel affinity, cation exchange, and size exclusion chromatography. Abiraterone was synthesized (Methods). Binding affinities were determined using a UV/vis spectral shift assay. Progesterone 17α-hydroxylation was evaluated using HPLC separation and UV detection. For crystallography, inhibitors were included throughout purification. Crystals were grown from CYP17A1 (30 mg/mL) complexed with inhibitor using hanging-drop vapor diffusion to equilibrate against 30% PEG 3350, 0.175 M Tris, pH 8.5, 0.30 M ammonium sulfate, and 3% glycerol. Diffraction data was collected and phased by molecular replacement. Iterative model building and refinement generated the final model. Substrates were docked using Surflex-Dock30 (link).