To study the five DNA gyrase mutations, a total of 231 alchemical free energies were calculated (8x λ simulations 0.5 ns long) and including equilibration simulations, a total of 1.17 µs of molecular dynamics simulations were initially performed. As described later, for DNA gyrase, nine calculations were extended to 5 ns which increased the total molecular dynamics performed to 1.49 µs. To avoid equilibration effects, the first 0.25 ns of each λ simulation was discarded. removing the electrical charge from atoms being perturbed, followed by transforming their van der Waals parameter, before finally recharging the atoms being perturbed. Double headed arrows represent the restraint used to prevent rifampicin from leaving the binding pocket. In all cases we are making use of the fact that free energy is a state function and therefore we can write the difference binding free energy (ΔΔG binding ) as a sum of so-called alchemical free energies (e.g. ΔG 4 -ΔG 3 ).