Hence the variation in DDG arises mainly from the apo and complexed values of DG vdW -the notable exceptions being gyrB E501D and gyrA D94G. This is despite our efforts to minimize the overall error by running up to 4x the number of repeats for those transitions (Table ) to reduce their individual estimated errors. For gyrB E501D and gyrA D94G all three transitions contribute significant error, which since they add in quadrature, leads to a large overall error in DDG. This is not surprising since both mutations involve turning off (and on) electrical charge and D94G involves a net charge change that must be compensated for elsewhere in the system. To investigate how far we might reduce the errors, let us now consider the individual values of DG qoff , DG vdW and DG qon (Figure ). By starting each simulation from a different structural seed and discarding the first half of the alchemical free energy simulations and then applying statistics to the resulting values of DG we are assuming that they are independent. If true, then one would also expect the values to be normally distributed which would appear to be the case for most sets of DG values (Figure ).