Structural basis of proton translocation and force generation in mitochondrial ATP synthase
- Max Planck Institute of Biophysics, Germany
Figures
Figure 1 with 3 supplements
Cryo-EM structure of the Polytomella sp.
F1 Fo ATP synthase dimer at 4.1 Å resolution. Subunit a, blue; c-ring, yellow; ASA 6, brick; other subunits, cyan; detergent micelle, grey.
Figure 1—figure supplement 1
Schematic diagram of ATP synthase dimer from mitochondria of Polytomella sp.
c-ring rotor, yellow; subunit a, dark blue; subunit ASA 6, brick; α and β subunits of catalytic F1 head, green; central stalk subunits, light blue; oligomycin-sensitivity conferring protein (OSCP), …
Figure 1—figure supplement 2
Cryo-EM of Polytomella sp.ATP synthase dimer.
(A) Electron micrograph of detergent-solubilised ATP synthase dimers. Scale bar, 500 Å; (B) Fourier shell correlation (FSC) curve and local resolution for the 1.6 MDa dimer indicate 4.1 Å overall …
Figure 1—figure supplement 3
Subunit a sequence alignment.
Strictly conserved aArg239, light blue; conserved charged and polar residues, pink; small residues aAla246, aGly247 and aAla292 at the point where the lumenal channel passes between H5 and H6, green;…
Figure 2 with 1 supplement
3.7 Å map of Polytomella Fo subcomplex with fitted atomic models.
(A) Side view with fitted subunit a, trans-membrane helices of ASA 6 and c-ring. (B) Subunit a and ASA 6 seen from the c-ring. (C) subunit a, c-ring and ASA 6 seen from the matrix. Subunit a, blue; c…
Figure 2—figure supplement 1
3.7 Å map with fitted atomic models.
Subunit a, blue; ASA 6, brick; c10-ring, yellow. The strictly conserved aArg239 in its well-defined map density is highlighted (blue ellipse).
Figure 3
Two aqueous channels in Fo.
(A) Lumenal channel seen from the crista lumen. (B) Matrix channel seen from the matrix. (C) Side view of both channels seen from the c-ring, with outer c-ring helices in transparent yellow. Lumenal …
Figure 4 with 3 supplements
Proton pathway through the Fo subcomplex.
(A) In the lumenal channel, protons (red arrow) pass via the local proton reservoir of aGlu172, aHis248, aHis252 and aGlu288 (dashed red ellipse) through the H5/H6 helix hairpin at the small …
Figure 4—figure supplement 1
Stereo diagrams of map density (blue mesh) with fitted atomic models.
(A) Lumenal channel passing through the H5/H6 helix hairpin at aAla246, aGly247 (H5) and aAla292 (H6) (green). (B) aArg239 is located halfway between the lumenal channel on the left and the matrix …
Figure 4—figure supplement 2
Stereo diagrams of bound lipid detergents.
(A) Lipid density modelled as two phosphatidyl glycerol molecules (grey) between subunit a (blue) and ASA 6 (brick); (B) detergent density modelled as dodecyl maltoside and hydrophobic alkyl chain …
Figure 4—figure supplement 3
Stereo diagrams of channel-lining residues seen from the protein interior.
(A) Lumenal channel; (B) matrix channel. Sidechains in stick representation are coloured as: subunit a, blue; subunit c-ring, yellow; ASA 6, brick. Channels are shown as potential surfaces (red, …
Figure 5 with 1 supplement
3.7 Å map of functionally important a-subunit residues with fitted atomic model.
(A) Proton reservoir formed by aGlu172, aHis248, aHis252, aGlu288 (dashed red ellipse) in the lumenal channel; (B) Interaction of aAsn243 (H5) and aGln295 (H6) stabilises the H5/H6 hairpin. The …
Figure 5—figure supplement 1
Disease-relevant residues in subunit a.
(A) Residues in a-subunit helices H4, H5 and H6, which are implicated in diseases are highlighted in orange. (B) Trp189 and Tyr299/Thr193 that act as wedges between H4 and H5.
Figure 6
c-ring rotation is powered by the potential gradient between the lumenal channel (pink) and matrix channel (light blue).
The c-ring (yellow) and the membrane-intrinsic four-helix bundle of subunit a (blue) drawn to scale as seen from the matrix. Protons (red) pass from the crista lumen below the projection plane …
Videos
Video 1
Three-dimensional map of the 1.6 MDa mitochondrial ATP synthase dimer from Polytomella sp.
showing the two c-rings (yellow), subunits a (blue) and ASA 6 (brick). The remaining eight ASA subunits in the peripheral stalks, the central stalks and catalytic F1 heads are shown in transparent …
Video 2
Three-dimensional arrangement of subunit a (blue), c-ring (yellow) with lumenal channel in pink and matrix channel in light blue.
https://doi.org/10.7554/eLife.33274.017
Video 3
Arrangement of channel-lining sidechains for the lumenal channel.
Sidechains in stick representation are coloured as: subunit a, blue; c-ring, yellow; ASA 6, brick; lipids, grey. Channels are shown as potential surfaces (red, negative; blue, positive; grey, …
Video 4
Arrangement of channel-lining sidechains for the matrix channel.
Sidechains in stick representation are coloured as: subunit a, blue; c-ring, yellow; ASA 6, brick. Channels are shown as potential surfaces (red, negative; blue, positive; grey, neutral).
Tables
Table 1
Cryo-EM data collection parameters, image processing, and refinement statistics.
https://doi.org/10.7554/eLife.33274.008| Data collection | |
|---|---|
| Electron Microscope | JEOL JEM-3200FSC |
| Camera | K2 Summit |
| Voltage | 300 kV |
| Energy filter slit width | 20 eV |
| Nominal Magnification | 30,000 x |
| Calibrated physical pixel size | 1.12 Å |
| Pixel size after mag. distortion corr. | 1.105 Å |
| Total exposure | 82.5 e-/Å2 |
| Exposure rate | 11.5 e-/(pixel x s) |
| Number of frames | 45 |
| Defocus range | −0.4 to −5 μm (95% between -0.9 and -2.5 µm) |
| Image Processing | |
| Motion correction software | Unblur/MotionCor2 (with mag. distortion corr.) |
| CTF estimation software | CTFFind4, Gctf (for per-particle CTF) |
| Particle selection software | e2boxer (EMAN2) |
| Micrographs used | 9,518 |
| Particles selected | 117,281 |
| 3D map classification and refinement software | Relion2 |
| Particles contributing to final map | 90,142 |
| Applied symmetry | C2 |
| Global resolution (FSC = 0.143) | 3.68 Å |
| Applied B-factor | -125 Å |
| Model Building | |
| Modeling software | Coot |
| Refinement software | Phenix (phenix.real_space_refine) |
| Number of residues built | 1,039 |
| RMS (bonds) | 0.01 Å |
| RMS (angles) | 1.09° |
| Ramachandran outliers | 0.0% |
| Ramachandran favoured | 94.88% |
| Rotamer outliers | 0.27% |
| Clashscore | 7.5 |
| EMRinger score | 1.42 |
