Figure 1. The structure of CI* from Vigna radiata.
(A) An overview of the conserved modular structure of CI using the Thermus thermophilus bacterial core subunits as a simple model (PDB: 4HEA) (Baradaran et al., 2013). (B) CryoEM density map of CI* from V. radiata highlighting its modular architecture. N, NADH-binding module; Q, quinone-binding module; PP, proximal-pump module; PD, distal-pump module; γCA, carbonic anhydrase domain, see also Video 1). (C) Atomic model of V. radiata CI* with all 30 assigned subunits labeled. The additional N-terminal helix of NDUS8 is indicated with an asterisk (*).
Figure 1—figure supplement 1. Schematic CI assembly pathways in metazoans and plants.
(A) CI assembly in metazoans. N-module is added at the last step of assembly. (B) CI assembly in plants. CI* intermediate is assembled before the PD domain is added last. N, NADH-binding module; Q, quinone-binding module; PP, proximal-pumps module; PD, distal-pumps module; γCA, carbonic anhydrase domain. Based on Formosa et al., 2018; Guerrero-Castillo et al., 2017; Garcia et al., 2017; Stroud et al., 2016; Ligas et al., 2019.
Figure 1—figure supplement 2. Purification and characterization of CI*.
(A-F) Representative preparation of CI* sample. (A) Digitonin-extracted, amphipol-stabilized mitochondrial membrane complexes were run on a 3–12% blue-native polyacrylamide gel electrophoresis (BN-PAGE) and subjected to in-gel NADH dehydrogenase activity assay to detect CI activity (purple bands). The band labeled Peak 2 corresponds to CI*. (B) The digitonin-extracted, amphipol-stabilized sample was separated by a 10–45% (w:v) linear sucrose (suc) gradient and fractionated. (C) Samples of the sucrose gradient fractions from (B) were run on BN-PAGE gels and subjected to in-gel NADH-dehydrogenase activity assay. Relevant fractions as indicated by the dashed boxes were separately pooled and concentrated. (D) The pooled fractions from (C) were furthered purified using size-exclusion chromatography. The trace for the absorbance at 280 nm is shown. Relevant peak fractions were pooled and concentrated. (E) The activity of the purified fractions from (D) was re-tested with an NADH-dehydrogenase in-gel activity assay. (F) The activity of the purified samples from (D) was further tested with a spectroscopic NADH-decylubiquinone (DQ) activity assay in the presence or absence of 100 µM DQ. Four independent repeat measurements were done for each sample. The background-corrected average of the repeats is shown, together with the standard deviation (error bars). Significance (**, p<0.01) was tested with a two-tailed t-test. p-values: 0.0005 (peak 2) and 0.0063 (peak 3). (G-H) Preparation of CI* for the cryoEM dataset presented in this paper. (G) The digitonin-extracted, amphipol-stabilized mitochondrial membrane complexes were separated by a 15–45% (w:v) linear sucrose gradient and fractionated. (H) Sucrose gradient fractions from (G) were subjected to in-gel NADH-dehydrogenase activity assay. Fractions 10–11 were pooled, concentrated, buffer-exchanged and used as the sample for the cryoEM grid used in the determination of the structure of CI* presented here.
Figure 1—figure supplement 3. CryoEM processing steps.
(A) A representative micrograph of the 8541 used for further processing (9816 collected). Scale bar, 100 nm. (B) Representative 2D class averages from reference-free classification in Relion. (C) Classification and refinement procedures used. The local refinement map, a local refinement slice and the gold-standard FSC curves are shown next to their respective final reconstructions.
Figure 1—figure supplement 4. CryoEM model-to-map correlation.
(A) Representative density from the composite map showing the fit between the model and thee map for elements of secondary structure from the peripheral and membrane arms of CI*. (B–D) Map-Model FSC curves are shown for the peripheral arm focused refinement (B), the membrane arm focused refinement (C) and the composite map (D). (E) Low contour of filtered cryoEM density for CI* colored by module (N, tan; Q, green; PP blue; and γCA pink). The lipid membrane density is shown in grey.