Extended Data Fig. 3: Conservation, covariance, and prior mutagenesis data mapped onto the stator complex structure.

a, Surface conservation as determined by Consurf⁵¹ (maroon high conservation, cyan low conservation). (Left) View from the cytoplasm showing conservation at the cytoplasmic MotA domains, including residues previously identified to be important for torque generation²⁵ (R90 and E98). (Centre) Side view showing poor conservation within membrane-interfacing residues of MotA. (Right) Cutaway displaying high level of conservation at the MotA-MotB interface; MotA shown as surface representation, MotB as ribbon. b–d, Evolutionary co-variation of residues (b) within MotA, (c) between MotA subunits, with boxes highlighting regions of strong covariance that are illustrated in more detail in the adjacent fragments, or (d) between MotA and MotB with the left hand side showing contacts for modelled MotB regions and the right hand side showing contacts in the unmodelled N-terminal MotB density represented here as a poly-alanine backbone. Predictions were carried out in Gremlin⁶ and contacts with a probability score of > 0.9 are shown. Contacts are coloured by Cα-Cα distance (≤10 Å in green, ≤15 Å in yellow). e, Mapping previous tryptophan scanning mutagenesis performed on MotA TMHs²³ (i) or MotB²⁴ (ii) to the stator complex structure. MotA and MotB are coloured as in Figs. 2 and 3 with targeted residues coloured according to toleration to mutagenesis; yellow corresponds to tolerated mutants (relative swarm rates > 0.5), red are poorly tolerated mutants (relative swarm rates of ≤ 0.5). In (i), TMH4 (green) and TMH2 (red) of neighbouring MotA subunits and MotB (white) are shown as transparent silhouettes. Poorly tolerated mutants cluster at subunit interfaces. In (ii) only TMH3-TMH4 of three MotA subunits are shown for clarity. f, Mapping previously determined cysteine crosslinks between (i) MotA-MotA²¹, (ii) MotB-MotB²², or (iii) MotA-MotB²¹ to our structure. For displaying crosslinks, a yield of ≥ 30% disulfide-linked adduct under iodine oxidizing conditions was used as threshold, except for MotA_TMH4-MotB crosslinks which used a ≥ 10% threshold. All analyses in this figure were performed using an E. coli MotAB structure generated by homology threading onto C. sporogenes MotAB.