In previous studies of S. aureus DHFR all alchemical free energies were repeated the same number of times which, since n values of the final difference in binding free energy (ΔΔG) were then obtained, simplified the calculation of errors. Both simulation unit cells studied here were over an order of magnitude larger and we therefore instead calculated errors at the level of an individual alchemical free energy (e.g. DG vdW ), with the final error in ΔΔG estimated by adding these in quadrature. Throughout a 95% confidence limit was estimated by multiplying the standard error by the appropriate t-statistic. We arbitrarily decided that at least three independent values of each alchemical free energy would be calculated, and then additional repeats would be run with the aim of reducing the magnitude of the overall 95% confidence limit to less than 1 kcal/mol. Achieving the latter was not always possible even when large numbers of repeats were run (n ≥ 10, see Supplementary Information).