Model building and refinements

Starting electrostatic potential maps were generated from the molecular replacement solution using rigid-body refinement in phenix.refine⁵¹. The starting model and maps were used for remodeling several side chains and manually placing the metal cofactor Zn²⁺ using Coot³⁷. After restrained reciprocal space refinement in phenix.refine, the resulting map was inspected for any difference potential, indicating the presence of the bound inhibitor. The AZM ligand was imported in Coot using the get monomer command, and we used the find ligands command to fit the ligand using the precalculated mFo–DFc difference potential map at 2.8σ.

Furthermore, upon inspection of the map, a clear blob of difference potential was observed in the solvent region, appropriate for accommodating a single DMSO molecule that was used as solvent to solubilize the ligand. The DMSO molecule was manually fitted using the get monomer command in Coot. Its position is in agreement with the neutron structure (PDB ID 4g0c)³⁰ where the space is occupied by three waters, and with a glycerol (GOL) solvent molecule occupying the same region in the high-resolution X-ray model (PDB ID 3hs4)²⁹.

The HCA II:AZM model was then completed, automatically placing water molecules, and using restrained reciprocal space refinement in phenix.refine⁵¹. All refinement steps were preformed using a test set representing 5% of all reflections, atomic scattering factors for electrons, automatic weighting of the experimental data to stereochemistry and atomic displacement parameter terms, and group B-factor refinement per residue⁵¹.