The simplest approach is to assume that a positive value of the change in binding free energy of the antibiotic (ΔΔG < 0) indicates that the antibiotic binds less well to the target following the mutation and therefore would be predicted to confer resistance to that drug. Clinically, however, a sample is categorized as 'resistant' if its minimum inhibitory concentration (MIC) is greater than a critical concentration, often the epidemiological cutoff value (ECOFF/ECV), which is defined as the MIC of the 99 th percentile of a collection of phenotypically-wildtype samples. Such thresholds for both drugs were derived using published ECOFF/ECV values 28 as described previously . [1] N is the total number of free energy calculations used to calculate the DDG, excluding the rpoB restraints as their contributions were negligible (see Supplementary Information) N min and N max list the minimum and maximum number of repeat calculations used for apo or drug-bound de-charging (DG qoff ), van der Waals (DG vdW ) or re-charging (DG qon ) transitions, respectively (Figure ). * indicates a gyrB mutation.