Overall, the accuracy of the optimized RBFE calculations were comparable to the benchmark results, regardless of the protocol employed. The most directly comparable result-a single simulation per mutation using Protocol C12-had an MAE of 1.05±0.08, with comparable results achieved with Protocols A, B, and C (See Table ). These values are on average slightly greater than the MAE and RMSE of 0.95 and 1.14 kcal/mol, respectively, reported by Song et al., but the difference in MAE is statistically insignificant for Protocols A, C, and C12 at the 95% confidence level. However, these results were achieved with over a 64% reduction in production computational cost. In the case of Protocol C, the average savings were over 85%. All protocols and all batch sizes achieved R 2 values greater than the 0.15 reported by Song et al. (see Table ).