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. 2021 Jan 19;10:e62047. doi: 10.7554/eLife.62047

Figure 1. CryoEM reconstructions for V. radiata mitochondrial CIII2, CIV and SCIII2+IV.

(A) CryoEM density map for CIII2 in isolation (not assembled into a supercomplex; see also Figure 1—figure supplement 1 and Video 1). (B) Density map for CIV, obtained from re-centered focused refinements of CIV in the supercomplex (see also Figure 1—figure supplement 2 and Video 2). (C) Composite map of SC III2+IV, assembled by combining CIII2 and CIV-focused refinements from the SC particles (see also Figure 1—figure supplements 1 and 2 and Video 3). Volume surfaces are colored by subunit (see also Videos 1–3 and Figures 2 and 5). The approximate position of the matrix and IMS sides of the membrane are shown with black lines.

Figure 1.

Figure 1—figure supplement 1. Initial processing and reconstructions using cryoSPARC.

Figure 1—figure supplement 1.

(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 of CIII2, SC III2+IV and CI* (last column). (C) Classification and refinement procedures used. Note that the same micrographs were used for the structural determination of V. radiata CIII2, CIV, SC III2+IV (this paper) and CI* (Maldonado et al., 2020) (green dashed box). The local resolution map and the half map gold-standard FSC curves are shown next to their respective final reconstructions.

Figure 1—figure supplement 2. Supercomplex focused classification and 3D refinement using Relion.

Figure 1—figure supplement 2.

SC III2+IV particles selected from 2D classification were first aligned using a SC III2+IV model. These aligned particles were sorted by several rounds of 3D classification using a CIV-only mask (see Materials and methods). Results from the 3D classifications were pooled and duplicate particles were removed. Particles from this set were re-extracted centered at CIII2 and CIV, generating two sets of re-centered particles. These were 3D-refined, CTF-refined and polished independently using a CIII2 and CIV mask/model, respectively. For the CIV-centered particles, an additional round of 3D classification was performed on the shiny particles. The local resolution map, a slice through the local resolution map and the half map gold-standard FSC curves are shown next to their respective final reconstructions. The locally refined maps were combined in Phenix to generate a composite map based on the best SC III2+IV reconstruction (bottom left inset; see also Figure 1—figure supplement 1).

Figure 1—figure supplement 3. Map-Model FSCs are shown for (A) CIII2 alone (see also Figure 1—figure supplement 1), (B) CIV-focused map from the supercomplex particles (see also Figure 1—figure supplement 2) and (C) the SC III2+IV composite map (see also Figure 1—figure supplement 2).

Figure 1—figure supplement 3.

See also Supplementary file 1b.

Figure 1—figure supplement 4. Fractionation and activity of extracted mitochondrial membranes.

Figure 1—figure supplement 4.

(A) Digitonin-extracted, amphipol-stabilized V. radiata mitochondrial membrane sample was separated by a 15–45% (w:v) linear sucrose (suc) gradient and fractionated. Relevant fractions were pooled and concentrated as indicated by dashed boxes and labels (fractions 4–8, 9–10, 11–13, 14–18) after the in-gel activity assay in (B). (B) Select fractions of the sucrose gradient fractions from (A) were run on a BN-PAGE gel and subjected to in-gel NADH-dehydrogenase activity assay. Note that not all fractions were loaded on the gel, and that the peak on fraction 18 corresponds to aggregation, likely due to the use of old mitochondrial samples due to COVID-19-related research restrictions. (C) The activity of the pooled samples from (A) was tested with a spectroscopic activity assay from reduced-decylubiquinone to cytochrome c, in the presence or absence of 2 µM antimycin (anti) or myxothiazol (myxo). Three to five independent repeat measurements were done for each sample. The background-corrected average of the repeats is shown, together with the standard error from the mean (S.E.M., error bars). Significance (**, p<0.01; ***, p<0.001) was tested with two-tailed t-tests for each inhibitor-exposed sample with respect to the control. p-values from left to right: 0.97, 0.32, 8.0 × 10−5, 1.5 × 10−4, 4.7 × 10−6, 2.8 × 10−5, 0.0064, 0.0051. (D) The activity of the pooled samples from (A) was tested with a spectroscopic activity assay to follow the oxidation of reduced cytochrome c, in the presence or absence of 4 µM potassium cyanide (KCN). Three to four independent repeat measurements were done for each sample. The background-corrected average of the repeats is shown, together with the standard error from the mean (S.E.M., error bars). Significance (*, p<0.05; ***, p<0.001) was tested with two-tailed t-tests for each inhibitor-exposed sample with respect to the control. p-values from left to right: 0.25, 0.0398, 0.0199, 7.9 × 10−4.