Fig. 5: Substantial cognate metalation predicted and confirmed in 4 mM manganese.

a Representative (n = 3 independent biological replicates) chromatogram showing in-cell acquired metals in MncA-containing fractions determined by ICP-MS, as in Fig. 4a, b but using a single anion exchange step (Q-Sepharose), HPLC SEC (TSK SW3000) and showing increased cognate metalation with Mn^II. MncA was identified by SDS-PAGE in Supplementary Fig. 10c. Metal contents of MncA-containing fractions (Supplementary Table 5) from independent biological replicates (Supplementary Fig. 10) were used to calculate fractional occupancies (%). b Metal availabilities (∆G_M, squares, and text inset) in E. coli cytosol grown aerobically as in Fig. 4c, except Mn^II replaced with estimates from calibrated qPCR of MntR target mntS in cells cultured in 4 mM manganese (Supplementary Fig. 4). MncA metal-preferences as ∆G_MP (pale blue circles, Cu^I, triangle). Bars are sensor ranges from 1% to 99%. Inset shows occupancies of MncA predicted from free energy gradients using Supplementary Data 4 and substituting Mn^II availability with a value of 4.5 × 10⁻⁵ M (as in inset text). The inset shows predicted MncA metalation (dark blue columns) with Mn^II, based on the largest favourable gradient (∆∆G_max in main figure), plus partial metalation with Fe^II. Inset also shows mean ( ± SD) in-cell metalation from the n = 3 independent biological replicates (square, circle, triangle, pale blue columns, as in Supplementary Table 5), largely matching predictions. Source data are provided as a Source Data file.