Two of the key decisions in ligand binding free energy calculations are the choices of the forcefield and how small molecules are parameterized. For simulations using Amber forcefields the choice of procedures for ligand preparation is usually whether to use AM1-BCC or Gaussian/RESP based protocols to determine atom charges in combination with the GAFF general purpose forcefield parameters. Following the choice in Wan et al. we used Gaussian/RESP for the majority of simulations in this work, but to evaluate the influence of this we re-ran the DIV dataset in the 2OSS model using the AM1-BCC methodology. Figure shows that the ∆G MMPBSA values for the large majority of the ligands are highly correlated between the two schemes (within 1-2 kcal mol -1 ). This and the similar correlation with experiment (shown in Table ) indicates that our results are robust with respect to this choice. The Wan et al. study employed the Amber ff99ildn forcefield for the protein, whilst in this study we have used ff14. In general the results obtained for all ligands are consistent but two ligands at either end of the rankings, THQ9 and THQ15, differ significantly as shown in Figure . The ranking performance with ff99ildn is described in Table . Comparing to those for ff14 (the THQ subset values in Table ) shows ff99ildn provides better results, especially those for 2traj-ar in the 4BJX model (r s of 0.80 compared to 0.46). There are many factors which may cause this difference but one we identified was the possibility that the balance between direct and water mediated interactions might be altered by modifications to the amino acid side chain parameters. This in part motivated our investigation of the impact of including explicit water molecules in the receptor component of our calculations (see the following section).