Ab initio phasing of salen ligand and biotin structures was performed using SHELXD or SHELXT from HKL files reduced by XDS . The resulting atomic coordinates were then further refined in ShelXLE as follows: Structures obtained from SHELXD/SHELXT were refined with a WGHT parameter of 0.2 over batches of 1000 cycles of least-squares refinement until the R-factors converged, using electron scattering factors parameterized as four gaussians for each element . Missing non-hydrogen atoms were assigned guided by Q-peaks in ShelXLE. Hydrogen atoms were placed when evident in the F o -F c map and Q-Peaks, and when appropriate based on likely molecular geometry, but were omitted during refinement for the purposes of comparison between datasets. All refinements were performed treating B-factors as isotropic. For each sample, representative structures were determined with riding hydrogens and anisotropic B-factor refinement whenever doing so did not result in non-positive definite (NPD) Bfactors on any atoms (Figure ). Structures of thiostrepton were determined by molecular replacement using MOLREP with PDB entry 1E9W as a search model and refined in PHENIX. Bond length restraints of 1.7 Å +/-0.02 Å were implemented for all five thiazole sulfur atoms and their neighboring backbone carbon atoms to keep the thiazole rings intact during cycles of refinement. Planarity restraints with a standard deviation of 0.005 Å were also applied over each atom in an sp 2 hybridization environment on the thiazole rings of the thiostrepton molecule. For each thiostrepton dataset used for structure determination, molecular replacement in MOLREP was also performed using the model from PDB entry 1E9W with all residues mutated to alanine , followed by a cycle of rigid body refinement in PHENIX , to visualize Fourier difference maps to reveal density for atoms not supplied in the search model. Isotropic B-factors were refined over residues.