Coordinate preparation and topology file generation. This package performs a number of steps to set up each FEP transformation. First, for every transformation (or edge) it aligns the coordinates of the two protein-ligand systems using OpenEye Spruce. Then it inserts the coordinates of ligand B into the coordinate file of the protein-ligand A complex, giving a coordinate file with both ligands present in the binding site. As a default, in a transformation from ligand A to ligand B the protein structure in complex with ligand A is used for the simulations, however, this can be easily adapted by the user to insert ligand A into the protein-ligand B complex. In a next step the script creates the topology files needed to perform all transformations in the thermodynamic cycle. Two topology files are generated to describe all transformations in the binding site. This is necessary due to our multi-step alchemical pathway that first perturbs some of the vdW interactions (Figure leg B), then the electrostatics (leg C) and finally the rest of the vdW (leg D). The first topology file describes leg B and leg C in the thermodynamic cycle and the second topology file describes the end states in leg D. The respective end states of the transformations were defined as an A and a B state in the [ moleculetype ] section of the combined ligands in the topology files. Below we will describe the details for generating the topology files. Example output topology files are provided in the SI.