Fig. 2: High persistence-conferring nuo* mutations target hydrophobic amino acids in transmembrane subunits and do not cause a complete loss of function of the complex.

a Modeled structure of E. coli complex I (see Methods). The cytoplasmic subunits NuoBCDEFGI (gray surfaces) catalyze NADH oxidation and transfer two electrons over a series of iron-sulfur clusters (not shown) to the quinone reduction site. The quinol flows further down the electron transport chain in the membrane (gray background) to be re-oxidized by terminal oxidases (not shown). The hydrophobic subunits NuoAHJKLMN (in colored surfaces) translocate four H⁺ ions per molecule of NADH that is oxidized by the cytoplasmic subunits. The membrane part is magnified in the inset and is annotated with the positions of amino acid variants found in high-persistence mutants (red spheres) and with residues that are crucial for proton translocation (blue sticks, based on refs. ^51,52,53). b, c High-persistence mutations are significantly enriched in b the membrane units and c predominantly target hydrophobic amino acids (Chi² comparison to random mutations, see Methods). d The nuo* mutations are causal for high persistence. Mutants with single mutations in each one of subunits L, M and N lose their high tolerance for amikacin (5h with 400 µg ml⁻¹) when nuo* mutations are repaired (mean ±stdevs, n = 3; *p < 0.0001 for a within-strain comparison from a two-way ANOVA with Šidák’s posttest). e Killing dynamics with amikacin (400 µg ml⁻¹) confirm the high persistence of nuo* point mutants in stationary phase and show that their effect cannot be mimicked by a single gene or operon knockout (in red). A model describing biphasic killing dynamics (lines ±95% CIs) was fitted to the data (means ±stdevs, n = 3; * fits are different based on AIC criterion). See also Supplementary Fig. 2. Source data are provided as a Source Data file.