To establish starting conformations for the MD-based ABFE calculations, ten distinct docked poses of the top-scoring chemical form for each compound in the Tier 1 and Tier 2 compound sets for each target were generated with the docking procedures described above. To make poses meaningfully different from each other, a new pose was accepted only when its root-mean-square deviations (RMSD) from all other poses were > 2Å or if any atom was > 5Å from the same atom in all other poses. Although these docked poses could in principle be used directly as starting points for the ABFE calculations, our pilot studies indicated that this yielded relatively poor results. We therefore pre-processed each pose with a short MD simulation of the protein-ligand complex using the same force field and explicit solvent as the subsequent free energy calculation (Section "Absolute binding free energy calculation"), thus allowing the system to equilibrate before the production calculations. In some cases, a docked pose was not stable, and the ligand moved away from the binding site during equilibration. Such poses were not advanced to the free energy simulation stage. Details of this MD equilibration step follow.