Fig. 3. Mutations that confer resistance to one drug are often sensitizing to other drugs.

a For each of the 122 identified mutants we calculated resistance to FEP, AZT, and PIP as the drug concentration that inhibits mutant growth, $\mathrm{IC}50_{\mathrm{AB}}^{\mathrm{Mut}}$, divided by that of the WT $\mathrm{IC}50_{\mathrm{AB}}^{\mathrm{WT}}$. The WT amino acid is marked with an X and mutations, which were not included in our design are marked in gray. Mutations which did not confer resistance to any drug were not enriched and could not be identified in our data are marked with a dot. The resistance level of the latter to all drugs is expected to be similar to the WT or smaller. Source data are provided as a Source Data file. b The inhibitory concentration of each mutant is plotted on a logarithmic scale for the different drugs to assess cross-resistance. WT inhibitory concentration is marked with a dashed line and a gray background represents our resolution as determined by drug dilution factor between measurements. c The predicted conformation of the three antibiotics within the Bla_ampC pocket. The antibiotics were docked using AutoDock Vina to the 3D crystal structure of Bla_ampC beta-lactamase (PDB 1KVL) with the S64G replacement to emulate the WT physico-chemical environment. The enzyme is shown in ribbon, and regions that contain residues that affect resistance are colored in the same colors as in 3a-b. Residues for which multiple substitutions confer antibiotic resistance are highlighted and shown in stick representation. Antibiotics are colored-coded by atoms (gray for C, red for O, blue for N, yellow for S, and white for H).