Following best practice , the free energies (Fig. ) required to remove the electrical charge on the perturbing atoms (DG qoff ), transmute the van der Waals parameters (DG vdW ) and recharge the remaining atoms (DG qon ) were separately calculated using GROMACS 2016.3 for RNAP and 2019.1 for DNAG. Each calculation required eight simulations at equally spaced values of the progress parameter, λ. To accelerate convergence, 10,000 replica exchanges were attempted between neighboring λ-simulations every 1,000 timesteps. The process was repeated for both apo and complexed forms of either the RNAP or DNAG, thereby resulting in six independent free energies (Fig. ). The timestep was reduced from 2 fs to 1 fs and LINCS constraints were removed for the vdW transitions for all DNA gyrase mutations and the qon transition of gyrA D94G to prevent crashing. To ensure the drug remained bound, a harmonic distance-based potential with spring constant 1,000 kJ mol -1 nm -2 was applied between the centers of mass of the drug and the RNAP beta subunit. Two additional free energies describing the cost of removing this restraint (Fig. ) were then also calculated.