Atomic structures of respiratory complex III2, complex IV, and supercomplex III2-IV from vascular plants
- Department of Molecular and Cellular Biology, University of California Davis, United States
- BIOEM Facility, University of California Davis, United States
Figures
Figure 1 with 4 supplements
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 …
Figure 1—figure supplement 1
Initial processing and reconstructions using cryoSPARC.
(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+…
Figure 1—figure supplement 2
Supercomplex focused classification and 3D refinement using Relion.
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 …
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).
See also Supplementary file 1b.
Figure 1—figure supplement 4
Fractionation and activity of extracted mitochondrial membranes.
(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 …
Figure 2 with 3 supplements
Overview of plant CIII2 atomic model.
(A) CIII2 in cartoon representation with co-factors in sphere representation. The approximate position of the inner mitochondrial membrane is shown with black lines, and the matrix and …
Figure 2—figure supplement 1
CIII subunit comparison.
(A-C) CIII subunits are shown for (A) V. radiata (this work), (B) S. cerevisiae (PDB: 6HU9) and (C) B. taurus (PDB: 1BGY). Yeast and bovine subunits were independently aligned with the corresponding …
Figure 2—figure supplement 2
Comparison of the cyt b conserved subunit (COB) in V. radiata (Vr), S. cerevisiae (Sc), Arabidopsis thaliana (At) and B. taurus (Bt).
(A) Sequence alignment of COB performed in Clustal Omega. Secondary-structure elements are highlighted in a rainbow pattern (starting with dark blue in the N-terminus to red in the C-terminus) and …
Figure 2—figure supplement 3
Comparison of select conserved and accessory subunits of CIII2 of V. radiata (Vr), S. cerevisiae (Sc), A. thaliana (At) and B. taurus (Bt).
(A,D) Superposition of cyt c1 (CYC1) (A) and Rieske iron-sulfur protein (UCR1) (D) subunits from V. radiata (teal), S. cerevisiae (dark pink, PDB 6HU9), B. taurus (light pink, PDB 1BGY). (B,E) …
Figure 3 with 2 supplements
V. radiata’s CIII2 mitochondrial processing peptidase (MPP) domain has a conserved architecture and active site but contains plant-specific secondary-structure elements not seen in other CIII-MPP subunits or in soluble MPP.
(A) Ribbon representation of the VrMPP-α (blue) and VrMPP-β (light blue) looking into the central cavity. Dashed rectangle indicates the location of the active site, detailed in (B). (B) MPP-β …
Figure 3—figure supplement 1
Further characterization of VrMPP subunits and their homologs.
(A-B) Electrostatic potential of surface of VrMPP-β (A) and VrMPP-α (B). Electrostatic potential was calculated using Delphi (Li et al., 2012; Li et al., 2019), with standard parameters. Red, …
Figure 3—figure supplement 2
Alignment of MPP-α homologs.
(A) Protein sequences of V. radiata‘s MPP-α subunit (VrMPPa), S. cerevisiae (Sc) and B. taurus (Bt) CIII2 (Cor2, UQCR2) and soluble MPP (MPPa) subunits were aligned with Clustal Omega. Key residues …
Figure 4
Conformational heterogeneity analysis of V. radiata CIII2 reveals complex-wide, coordinated movements and shows the swinging motion of UCR1 in the absence of substrates or inhibitors.
(A–B) CIII2-wide motions revealed by principal component 2 (A) and 3 (B). Frame 1 (left) and frame 20 (right) of the continuous motion of CIII2 (teal surface) are shown. Black arrows indicate the …
Figure 5 with 1 supplement
Overview of V. radiata CIV and its co-factors.
(A) CIV in cartoon representation colored by subunit with co-factors in sphere representation colored by atom. The position of the inner mitochondrial membrane is indicated with black lines, and …
Figure 5—figure supplement 1
Comparison of the COX1-3 subunits in V. radiata (Vr), S. cerevisiae (Sc), A. thaliana (At) and B. taurus (Bt).
(A,D,G) Superposition of COX1 (A), COX2 (D) and COX3 (G) subunits from V. radiata (teal), S. cerevisiae (dark pink, PDB 6HU9), B. taurus (light pink, PDB 5B1A). (B,E,H) Sequence identity percentages …
Figure 6 with 1 supplement
Subunit differences in V. radiata CIV.
(A–C) Superposition of subunits of V. radiata (Vr, teal), S. cerevisiae (Sc, dark pink; PDB: 6HU9) and B. taurus (Bt, light pink; PDB: 5B1A) CIV. Subunits were aligned with the corresponding V. …
Figure 6—figure supplement 1
CIV subunit comparison.
(A-C) CIV subunits are shown for (A) V. radiata (this work), (B) S. cerevisiae (PDB: 6HU9) and (C) B. taurus (PDB: 5B1A). Yeast and bovine subunits were independently aligned with the corresponding V…
Figure 7 with 1 supplement
Proton transfer pathways of V. radiata CIV.
VrCOX1 (transparent ribbon), co-factors (stick) and key residues (colored stick) are shown for the D channel (yellow), K channel (purple) and H channel (green). Proton-channel residues that are …
Figure 7—figure supplement 1
Sequence alignment of COX1 highlighting the H, D and K channels.
COX1 sequences from V. radiata (Vr), Arabidopsis thaliana (At), S. cerevisiae (Sc), and B. taurus (Bt) were aligned with Clustal Omega. Proton pathway residues are highlighted: D channel in yellow, …
Figure 8 with 1 supplement
SCIII2+IV interface in V. radiata.
(A) General orientation of SC III2+IV in ribbon representation viewed from the membrane. Approximate position of the inner mitochondrial membrane is shown. Sites 1 (S1) and 2 (site 2) of the …
Figure 8—figure supplement 1
Differences in SC III2+IV interactions between V. radiata and S. cerevisiae (PDB: 6HU9).
The supercomplexes are aligned by V. radiata’s COB and CYC1 and shown in surface representation. (A-B) V. radiata (A) and S. cerevisiae (B) SC III2+IV viewed from the membrane. (C-D) Superposed V. …
Videos
Video 1
CryoEM density map and model for V. radiata CIII2.
Video 2
CryoEM density map and model for V. radiata CIV.
Video 3
CryoEM density map and model for V. radiata SC III2+IV.
Video 4
Superposition of V. radiata MPP-β with S. cerevisiae Cor1 (6HU9) and soluble MPP-β (1HR6).
Video 5
Superposition of V. radiata MPP-α with S. cerevisiae Cor2 (6HU9) and soluble MPP-α (1HR6).
Video 6
3D variability analysis of V. radiata CIII2, component 0.
The 3DVA volumes are shown as a continuous movie. CIII2 in teal, QCR10 in dark purple.
Video 7
3D variability analysis of V. radiata CIII2, component 1.
The 3DVA volumes are shown as a continuous movie. CIII2 in teal, QCR9 in lilac, QCR10 in dark purple.
Video 8
3D variability analysis of V. radiata CIII2, component 2.
The 3DVA volumes are shown as a continuous movie. CIII2 in teal.
Video 9
3D variability analysis of V. radiata CIII2, component 3.
The 3DVA volumes are shown as a continuous movie. CIII2 in teal.
Video 10
Swinging motion of the V. radiata UCR1 head domains.
The 3DVA volumes are shown as a continuous movie. A V. radiata UCR1 head-domain homology model was rigid-body fit into the 3DVA volume.
Tables
Table 1
Cryo-EM data collection, reconstruction, model refinement and validation statistics.
| Data Collection and processing | ||||||||
|---|---|---|---|---|---|---|---|---|
| Microscope | Titan krios (UCSF) | |||||||
| Camera | K3 detector equipped with GIF | |||||||
| Magnification | 60,010 | |||||||
| Voltage (kV) | 300 kV | |||||||
| Electron exposure (e-/Å2) | 51 | |||||||
| Defocus range (µm) | −0.5 to −2.0 | |||||||
| Pixel size (Å) | 0.8332 | |||||||
| Software | SerialEM | |||||||
| Reconstruction | CIII2 | SCIII2+IV | CIII2 focused | CIV-focused | SC Composite | |||
| Software | cryoSPARC | cryoSPARC | Relion | Relion | Phenix | |||
| Number of particles | 48,111 | 28,020 | 38,410 | 29,348 | --- | |||
| Box size (pixels) | 512 | 512 | 512 | 512 | 512 | |||
| Final resolution (Å) | 3.2 | 3.8 | 3.7 | 3.8 | --- | |||
| Map sharpening B factor (Å2) | 67 | 61 | 83 | 77 | --- | |||
| EMDB ID | 22445 | 22449 | 22450 | 22447 | 22448 | |||
| Model | CIII2 | CIV | SC composite | |||||
| Software | Phenix | |||||||
| Initial model (PDB code) | 6Q9E, 6HU9 | 6HU9, 5B1A | 6Q9E, 6HU9, 5B1A | |||||
| Map/model correlation | ||||||||
| Model resolution (Å) | 3.3 | 3.9 | 3.9 | |||||
| d99 (Å) | 3.5 | 3.9 | 3.9 | |||||
| FSC model 0.5 (Å) | 3.3 | 3.8 | 3.8 | |||||
| Map CC (around atoms) | 0.88 | 0.85 | 0.84 | |||||
| Model composition | ||||||||
| Non-hydrogen atoms | 32,931 | 12,772 | 45,164 | |||||
| Protein residues | 3983 | 1497 | 5472 | |||||
| Number of chains | 20 | 10 | 30 | |||||
| Number of ligands and cofactors | 8 | 6 | 14 | |||||
| Number of lipids | 29 | 20 | 43 | |||||
| Atomic Displacement Parameters (ADP) | ||||||||
| Protein average (Å2) | 114.37 | 38.28 | 53.17 | |||||
| Ligand average (Å2) | 79.11 | 50.71 | 66.25 | |||||
| R.m.s. deviations | ||||||||
| Bond lengths (Å) | 0.005 | 0.006 | 0.007 | |||||
| Bond angles (°) | 0.704 | 0.853 | 1.107 | |||||
| Ramachandran Plot | ||||||||
| Favored (%) | 93.13 | 90.75 | 92.55 | |||||
| Allowed (%) | 6.82 | 9.18 | 7.40 | |||||
| Disallowed (%) | 0.05 | 0.07 | 0.06 | |||||
| Validation | ||||||||
| MolProbity score | 1.97 | 2.19 | 2.04 | |||||
| Clash score | 10.03 | 14.21 | 11.51 | |||||
| Rotamer outliers (%) | 0.03 | 0.08 | 0.04 | |||||
| EMRinger score | 2.80 | 2.32 | 2.01 | |||||
| PDB ID | 7JRG | 7JRO | 7JRP | |||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Vigna radiata) | V. radiata seeds | Todd’s Tactical Group | TS-229 | Lot SMU2-8HR; DOB 2/25/2019 |
| Chemical compound, drug | Digitonin, high purity | EMD Millipore | 300410 | |
| Chemical compound, drug | A8-35 | Anatrace | A835 | |
| Chemical compound, drug | Gamma-cyclodextrin | EMD Millipore | C4892 | |
| Chemical compound, drug | Decylubiquinone | Santa Cruz Biotechnology | sc-358659 | |
| Chemical compound, drug | Equine cytochrome c | Sigma Aldrich | C2506 | |
| Software, algorithm | Clustal Omega | Madeira et al., 2019 | RRID:SCR_001591 | |
| Software, algorithm | Geneious | Kearse et al., 2012 | RRID:SCR_010519 | |
| Software, algorithm | SerialEM | University of Colorado, Schorb et al., 2019 | RRID:SCR_017293 | |
| Software, algorithm | RELION 3.0 | Zivanov et al., 2018 | RRID:SCR_016274 | |
| Software, algorithm | Motioncor2 | Zheng et al., 2017 | ||
| Software, algorithm | Ctffind4 | Rohou and Grigorieff, 2015 | RRID:SCR_016732 | |
| Software, algorithm | crYOLO | Wagner et al., 2019; Wagner and Raunser, 2020 | RRID:SCR_016732 | |
| Software, algorithm | Phyre2 | Kelley et al., 2015 | ||
| Software, algorithm | Coot | Emsley and Cowtan, 2004 | RRID:SCR_014222 | |
| Software, algorithm | PHENIX | Liebschner et al., 2019; Goddard et al., 2018; Pettersen et al., 2004 | RRID:SCR_014224 | |
| Software, algorithm | UCSF Chimera | Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, Pettersen et al., 2004 | RRID:SCR_004097 | |
| Software, algorithm | UCSF ChimeraX | Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, Goddard et al., 2018 | RRID:SCR_015872 | |
| Software, algorithm | PyMOL Molecular Graphics System | Schrödinger, LLC | RRID:SCR_000305 | Version 2.0 |
| Software, algorithm | Scaffold Proteome Software | Proteome Software Inc | RRID:SCR_014345 | Version 4.8.4 |
| Software, algorithm | X! Tandem | The GPM | Version Alanine (2017.2.1.4) | |
| Other | Holey carbon grids | Quantifoil | Q310CR1.3 | 1.2/1.3 300 mesh |
Additional files
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Supplementary file 1
(a) Mass spectrometry identification of V. radiata CIII2 and CIV subunits. (b) Model-building statistics by subunit. (c) CIII2 and CIV subunit homologs in plants, yeast and mammals. V. radiata homologs were obtained by performing BLASTp searches of the Arabidopsis thaliana genes (Meyer et al., 2019). Mammalian and yeast homologs were obtained from Hartley et al., 2019; Maréchal et al., 2012. Additional BLASTp searches were performed as needed. (d) RNA edits identified in V. radiata CIII2 and CIV subunits. RNA-editing sites were initially identified by an unambiguous mismatch in the cryoEM density and the expected density for the mitochondrial-DNA-encoded residue. The existence of the RNA-editing site in other plants, or the implied restoration of the consensus sequence was then inspected. Amino-acid changes to the atomic model (with respect to the mt-DNA sequence) were only made for amino-acid positions that had unambiguous cryoEM density evidence and whose editing is conserved or would restore the conserved sequence.
- https://cdn.elifesciences.org/articles/62047/elife-62047-supp1-v1.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/62047/elife-62047-transrepform-v1.docx
