Protein and ligand pre-processing separates the protein and ligand into separate docking-ready structures. Initially, a set of co-crystallized protein-ligand structures for each receptor in the Similarity Dataset is downloaded from RCSB.org . The ligand of interest in each co-crystallized structure is then designated based on its residue name. Using ProDy , separate files are generated for the protein, ligand, waters, and other heteroatoms that are present in the original crystal structure. The instance coordinates of the ligand structure are downloaded directly from RCSB.org using their File Download Service to ensure proper coordinate and bond information. Using only the protein and ligand complex, we align all structures of a receptor based on the residues closest to the binding pocket. Protein residues within 15 Åof the ligand are used to align each receptor structure with PyMol to the alphabetically lowest PDB ID (PDB IDs specified in Table ??). The co-crystallized ligand's coordinates are transformed according to the receptor alignment object. The aligned ligand and receptor are then separated into their own files to provide a ground-truth ligand and a holo structure for cross-docking with non-cognate ligands. After removal of the ligand, missing residues, atoms, and hydrogens are added (at a pH of 7.0) to the receptor structures and minimized, ensuring interactions are detected properly after docking. Finally, a gnina docking command template is created for each protein. The gnina docking command template specifies the docking protein structure (i.e. the aligned-to receptor structure) as well as the binding box location, defined by the cognate ligand of the docking receptor.