Figure 1. Overall structure of the Mrp complex.

(a) Cryo-EM density of a monomer, coloured by subunit as indicated. (b) Cryo-EM density of a dimer, with the model shown as a cartoon. (c) Model of the dimer, with each subunit coloured differently. Side view and view from the cytoplasm, where two N-terminal helices of subunit MrpE, forming most of the dimer interface, are circled. (d) Schematic view of the Mrp monomer, MBH complex and complex I. Homologs of Mrp subunits are coloured similarly as in c), with an additional MrpD-like subunit in complex I in orange.

Figure 1—figure supplement 1. Purification and characterization of Anoxybacillus flavithermus Mrp.

(a) Operon encoding seven subunits (MrpA-MrpG) of Anoxybacillus flavithermus WK1 mrp. Two modules based on the Mrp structure are indicated. (b) Mrp was purified by affinity- and size exclusion chromatography as a final step. The size exclusion elution profile is shown, with LMNG as detergent. The fraction in the middle of the dimer peak (which eluted at around 12.5 ml) was diluted for cryo-EM sample preparation. The elution peaks of the dimer and monomer are highlighted in blue and green, respectively. SDS-PAGE shows the presence of all Mrp subunits, identified by mass-spectrometry. (c) Size exclusion chromatography of Mrp in two different detergents - DDM and LMNG. The elution of the dimer is highlighted in blue. (d) Size exclusion chromatography of non-mutated (AF_Mrp) and mutated Mrp (MrpE L41W and MrpF D35L). The elution peaks of the dimer and monomer are highlighted in blue and green, respectively. (e) SDS-PAGE of the size exclusion chromatography fractions for the mutants MrpF D35L and MrpE L41W shows that all subunits co-elute in a single peak in each case. (f) Na⁺/H⁺ antiport activity of AF_Mrp in everted membrane vesicles (EMV) using NaCl to initiate Mrp activity at pH 7.5, pH 8.5 and pH 9.5. Non-transformed KNabc vesicles were used as a control at pH 8.5. The addition of succinate, NaCl and NH₄Cl is indicated with arrows. The 100% percentage of acridine orange fluorescence dequenching is shown when 10 mM NH₄Cl was added. (g) Na⁺/H⁺ antiport activity of AF_Mrp in EMV using KCl to initiate Mrp activity at pH 7.5 and pH 8.5. Non-transformed KNabc vesicles were used as a control at pH 8.5. (h) Na⁺/H⁺ antiport activity of AF_Mrp using NaCl to initiate Mrp activity is shown as the percentage of dequenching at pH 6.0, pH 7.5, pH 8.5 and pH 9.5. The results are averages from three independent preparations. The error bars show the standard deviations of the means. (i) Schematic of the Na⁺/H⁺ antiport activity using EMV. 1 – Addition of succinate to initiate respiration. 2- Oxidases create a ΔpH. 3 – Quenching of acridine orange. 4- Addition of NaCl to initiate Mrp activity. 5 – Dequenching of acridine orange, which is a measure of the Na⁺/H⁺ activity. 6 – Addition of NH₄Cl to establish a baseline. (j) Experiments of complementation capacity of AF_Mrp and AF_Mrp mutants MrpF D35L, MrpG S72W, MrpE L41W for the growth of sodium sensitive E. coli KNabc cells. Non-transformed KNabc cells were used as a control. The results are averages from three independent experiments. The error bars show the standard deviations of the means.

Figure 1—figure supplement 2. Processing of the Mrp-DDM dataset.

Representative raw micrograph is shown with some of picked particles circled. After classification, the two best classes were either further classified to process the Mrp dimer, or symmetry expanded according to the C2 point group to process the Mrp monomer. Further 3D classifications and 3D auto-refinements resulted in three different dimer classes with distinct angles between the monomers of the dimer and indicated resolutions. 3D auto-refinement, 3D classification, post processing and polishing of the symmetry expanded particles resulted in a reconstructed monomer map with a final resolution of 3.41 Å.

Figure 1—figure supplement 3. Processing of the Mrp-LMNG dataset.

Representative raw micrograph is shown with some of picked particles circled. After classification, the best class was either further classified to process the Mrp dimer, or 3D auto-refined and symmetry expanded according to the C2 point group to process the Mrp monomer. 3D classification to process Mrp dimer resulted in six classes with distinct angles between the monomers. 3D auto-refinement of the best class in C2 symmetry, resulted in a reconstructed dimer map with a resolution of 3.7 Å. The symmetry expanded particles were further classified to process the Mrp monomer, resulting in two good classes. 3D auto-refinement, post processing and polishing resulted in a reconstructed Mrp monomer map with a resolution of 2.98 Å.

Figure 1—figure supplement 4. Examples of cryo-EM density of Mrp monomer in LMNG.

(a) Densities of transmembrane helices from MrpA-G. (b) Densities of β-sheets from MrpA, MrpD and MrpE. (c) Density of phosphatidylethanolamine.