Structure Determination of mitoNEET

Purified mitoNEET protein (20 mg/ml) mixed with an equal molar concentration of furosemide was screened by sitting drop vapor diffusion against Index screen (Hampton Research). Red crystals formed in under a week in several conditions. Final data collection was performed using crystals grown by sitting drop vapor diffusion by mixing a 1:1 ratio of protein with 60% v/v Tacsimate pH 7.0. The mother liquor was used for cryoprotection. Data collection was performed on the Northeastern Collaborative Access Team beamline 24-ID-C at the Advanced Photon Source at the iron peak. Data were indexed, integrated, and scaled using iMOSFLMand Aimless. To avoid model bias molecular replacement was combined with anomalous signals (MR-SAD) using Phaser and PDB 3REE as the search model^{15 (link)}. Refinement was performed using phenix²⁷ . Model building/rebuilding was performed using Coot^{28 (link)}. OMIT maps were constructed by removing the ligand from the final refined structure file, performing three macro-cycles of refinement with simulated annealing, and calculating a |F_o|—|F_c| map with phenix.maps.