(A) SDS-PAGE depicting the sample solubilized in detergent after affinity chromatography (DDM) and the sample reconstituted into lipid nanodiscs after size-exclusion chromatography (ND). (B) Size-exclusion chromatography profile after complex I reconstitution into lipid nanodiscs. The main peak at 13 mL consists mostly of the intact reconstituted complex I. Fractions marked by the green, dashed rectangle were pooled, concentrated, and used for cryo-EM and activity assays. The second peak (retention volume 15 mL and higher) contains empty nanodiscs and the dissociated peripheral arm. (C) Mass photometry of the reconstituted complex I pooled form the main gel filtration peak. Left: a representative mass histogram showing two main peaks: ‘Empty’ nanodiscs at 95 kDa that co-elutes with complex I during size-exclusion chromatography and the nanodisc-reconstituted complex I at 713 kDa. (D) Values of Vmax for the spectrophotometric activity assays: NADH:Q1 (2.2±0.2 µmol NADH min−1 mg−1), NADH:Q1 inhibited with 20 µM piericidin A (0.4±0.1 µmol NADH min−1 mg−1), NADH:DQ (5±1 µmol NADH min−1 mg−1) and NADH:DQ inhibited with piericidin A (0.3 µmol NADH min−1 mg−1). The NADH:FeCy Vmax is not shown (117±7 µmol NADH min−1 mg−1). The graph shows mean ± SD. Results of individual measurement are shown by dots. (E) Representative traces of the NADH:FeCy (left), NADH:Q1 (middle, solid line), and NADH:DQ (right, solid line) activities. The NADH:Q1 and NADH:DQ inhibition assays are shown as dashed lines. Piericidin A addition point is marked as ‘+PA’. The concentration of complex I was 10 times lower for NADH:FeCy compared to that of NADH:Q1 and NADH:DQ assays.