Using the same input structures and parameters as in the previous study by Ge et al. allows for a comparison of SepTop and traditional RBFE in specifically the efficiency of the free energy perturbation independent from the effect of the force field or system preparation. The ligands were parameterized with Open Force Field version 1.0.0 (codenamed "Parsley") and AM1-BCC charges. The AMBER ff99sb*ILDN force field was used to parameterize the protein and the TIP3P model was used for the water. GROMACS was used to solvate the ligands and the protein-ligand systems and to add ions to reach a salt concentration of 150 mM. The output topology and coordinate files were then used to create the input files for SepTop. For the ERα system, we ran calculations using structures prepared in three different ways. We prepared the first structure ourselves using a protocol we detail below. The other two input structures were used in the studies of Azimi et al. and Zou et al.. and were provided by the authors. These systems were parameterized using Gaff1.8, 46 Amber ff14SB, and TIP3P water. To prepare our own structure, we began with PDB 2Q70. We performed the structure preparation on OpenEye's Orion cloud computing platform, using their workflow ("floe") "SPRUCE -Protein Preparation from PDB Codes" with the default parameters. In that workflow hydrogen atoms were added, missing loops were built and crystallographic waters were retained. The two chains of the homodimer were separated and chain A was used for the rest of the study. The binding mode and coordinates of the ligands were obtained from the SI of Zou et al. The prepared protein was aligned onto the protein provided by Zou et al. to be in the correct reference frame for the ligand coordinates. Orion floes "Bound Protein-Ligand MD" for the proteinligand complex and "Solvate and Run MD" for the unbound ligands were used to solvate, pa-rameterize, and equilibrate the systems. Ions were added to each to achieve a salt concentration of 50 mM. The GAFF1.81 force field and AM1-BCC charges were used for the ligands, Amber ff14SB parameters for the protein, and TIP3P 44 for the water. In the Orion floes, the systems were energy minimized, equilibrated and then a production run of 2 ns was performed. The trajectory of that simulation was then used to create the input files for Sep-Top.