Extended Data Fig. 4: Comparison of albicidin, FQ and gepotidacin binding sites.

Gyr-Mu217-albicidin structure is shown as cartoon representation with albicidin shown as yellow sticks. GyrA is shown in beige, DNA in teal and GyrB in coral. Catalytic Tyr122 and parts of GyrB are not shown for clarity. Albicidin residues, GyrA/GyrA’ interface (α3/α3’) and DNA bases next to the break (+1/-1) are indicated. To compare binding sites of different compounds, E. coli gyrase-gepotidacin cryo-EM structure (PDB:6RKS) and moxifloxacin (MFX) - Staphylococcus aureus crystal structure (PDB:5CDQ) were superimposed onto Gyr-Mu217-albicidin and aligned to the GyrA’ protomer using match maker in ChimeraX⁷¹. Two MFX molecules (MFX, MFX’) are shown as orange stick representations. A single bound gepotidacin molecule is shown as magenta sticks, with DNA-binding left-hand-side (LHS) and hydrophobic pocket binding right-hand-side (RHS) labelled. Note the overlap between the gepotidacin RHS and the methoxy group of pMBA5. MFX is bound via a so called ‘water–metal ion bridge’ to Ser83 and Asp87. The distance between the Asp87 and MFX in one protomer versus another illustrates large-scale movement of the albicidin-bound gyrase in comparison to the moxifloxacin-bound structure.