The protonation states of the protein’s ionizable groups are predetermined by the user-selected residue templates. Protonation states of the ligand are set with the program OpenBabel45, for a pH chosen using the ligand_ph variable. The current BAT software automates free energy calculations using any AMBER protein force field, chosen with the receptor_ff variable. Ligand parameters are assigned with the AM1-BCC charge model51 and either version 1 or 2 of the General AMBER Force Field (GAFF)52,53, chosen with the ligand_ff input variable. Currently supported options for the water model, chosen with the water_model parameter, are TIP3P54, TIP4PEw55 or SPC/E56. The types of any dissolved ions are specified with the cation and anion input variables, and the ions are assigned Joung and Cheatham parameters appropriate to the selected water model57. This selection could easily be changed by modification of the Python code.

The Ambertools tleap software42,43 is used to solvate the protein-ligand complex and the dummy atoms in a box of water molecules. The code allows the user to choose the number of water molecules in the box and the water padding in the x and y axes, using the num_waters, buffer_x and buffer_y parameters, respectively. The dependent variable is the water padding in the z direction, which is calculated using an efficient iterative relaxed Newton-Raphson approach, based on the cross-sectional xy area of the box, the requested number of water molecules, and the average tleap atomic density for each water model. With the neutralize_only keyword, dissolved counterions are added if needed for charge neutralization. With the num_cations parameter, a number of additional cations are also added for a desired ionic strength, with the same number of anions added for neutrality.

The binding free energy calculations also require simulations of the free protein and the free ligand. These calculations are automatically set up as follows. The size of the ligand box is set in the input file using the lig_buffer parameter; this defines the water padding in all three Cartesian axes. Counterions are added to neutralize the ligand, as needed. The variable num_cat_ligbox sets a user-defined number of additional cations, and the number of anions again is the dependent variable. The variables to create the box with the apo protein are the same used previously for the protein-ligand system, such as the number of waters, water model, x and y buffering, and number of cations.

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