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Atomic structure of the entire mammalian mitochondrial complex I

Nature volume 538, pages 406–410 (2016)Cite this article

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Abstract

Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane1,2. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons3. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active–deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.

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Figure 1: Structure of ovine complex I.
Figure 2: Arrangement of redox centres and substrate binding sites.
Figure 3: Additional cofactors identified in the structure.
Figure 4: Mechanism of mitochondrial complex I.

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Data deposits

The electron microscopy maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-4084, EMD-4090, EMD-4091 and EMD-4093 (combined map). The model has been deposited in the Protein Data Bank (PDB) under accession code 5LNK.

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Acknowledgements

We thank the ETH Zurich ScopeM Center for access to Titan Krios EM. Data processing was performed at the IST high-performance computer cluster. K.F. is partially funded by the Medical Research Council UK PhD fellowship. J.A.L. holds a long-term fellowship from FEBS. This project has received funding from the European Union’s 2020 research and innovation programme under grant agreement No 701309.

Author information

Authors and Affiliations

  1. Institute of Science and Technology Austria, Klosterneuburg, 3400, Austria

    Karol Fiedorczuk, James A. Letts, Karol Kaszuba & Leonid A. Sazanov

  2. MRC Mitochondrial Biology Unit, Cambridge, CB2 0XY, UK

    Karol Fiedorczuk

  3. MRC Laboratory of Molecular Biology, Cambridge, CB2 OQH, UK

    Gianluca Degliesposti & Mark Skehel

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  1. Karol Fiedorczuk
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Contributions

K.F. purified complex I for grid preparation, prepared cryo-EM grids, acquired and processed EM data, and co-built the models; J.A.L. purified complex I for cross-linking experiments, analysed cross-linking data and co-built the models; G.D. performed cross-linking/mass-spectrometry experiments, K.K. performed model re-building in Rosetta and sequence alignments; G.D. and M.S. analysed cross-linking data; L.A.S. designed and supervised the project, processed and analysed data and wrote the manuscript, with contributions from all authors.

Corresponding author

Correspondence to Leonid A. Sazanov.

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Reviewer Information

Nature thanks P. Ädelroth, M. Ryan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Image processing procedures.

a, Representative micrograph of 2.6 k micrographs collected that all varied in defocus, ice thickness and particle count, with good quality particles circled. Scale bar, 100 nm. b, Representative 2D class averages obtained from reference-free classification. c, Classification and refinement procedures used in this study.

Extended Data Figure 2 Image and model refinement procedures.

a, Radiation-damage weighting. Relative B-factors (Bf) and intercepts (Cf) from the Relion particle polishing procedure. b, Left, gold-standard (two halves of data refined independently) Fourier shell correlation (FSC) curves for the maps of the entire complex complete map (resolution at FSC = 0.143 is 3.9 Å), membrane-arm-focused refinement (4.1 Å resolution) and peripheral-arm-focused refinement (3.9 Å resolution). Right, FSC curve of the combined map versus final model shows good agreement of the model with the map (FSC = 0.5 at 4.0 Å resolution). FSC curve against the entire complex complete map, which was not used in refinement, is shown as a control. c, Statistics of refinement.

Extended Data Figure 3 Local resolution estimation and combination of maps for model building.

a–c, Local resolution estimation by Resmap of the entire complex I (a), peripheral-arm-focused refinement map (b) and membrane-arm-focused refinement map (c). Maps are coloured according to the shown resolution scale in Å. d. The final map was produced by combining maps with the best local resolution features; that is, for peripheral-arm-focused refinement map (orange), for the distal part of membrane-arm-focused refinement map (green), for 42-kDa subunit map from the selected homogenous complex I class (64 k particles; blue) and the rest of the complex from the best map of the entire complex (magenta).

Extended Data Figure 4 Examples of cryo-EM density.

a, b, Coils and α-helices from core (a) and supernumerary (b) subunits. c, d, Example β-sheets from core PSST subunit (c) and supernumerary 39-kDa subunit (d). Cryo-EM density is shown with the model represented as sticks and coloured by atom with carbon in grey, oxygen in red, nitrogen in blue and sulfur in yellow.

Extended Data Figure 5 Identified cross-links.

a, Solvent-accessible surface (SAS) representation of cross-links. Surfaces for complex I subunits are shown transparent and coloured as in Fig. 1. Shortest SAS paths calculated using Xwalk54 are shown for cross-links as coloured worms with inter-subunit lysine reactive cross-links in blue, inter-subunit acid reactive cross-links in red, intra-subunit lysine reactive cross-links in light blue, intra-subunit acid reactive cross-links in light red. b, Inter-subunit cross-link schematic. Complex I subunits are shown in a similar orientation as in a. Left panel with core subunits cyan, previously assigned supernumerary subunits in magenta, newly assigned or newly built regions of supernumerary subunits in green, poly-alanine regions in orange and unmodelled regions in red. Observed cross-links are indicated by dashed black lines between either blue circles (lysine reactive cross-links) or red circles (acid reactive cross-links). No cross-links were observed to the core subunits of the membrane arm and hence they were omitted for clarity. The horizontal black lines indicate the approximate boundaries of the inner mitochondrial membrane. Subunits B14.7, B15 and ASHI are shown as being behind the membrane boundaries as they are found on the opposite (far) side of the membrane arm.

Extended Data Figure 6 Folds of supernumerary subunits.

Subunits are shown in cartoon representation, coloured blue to red from N to C terminus. Disulfide bridges are shown as sticks with sulfur in yellow.

Extended Data Figure 7 Examples of supernumerary subunits interactions.

a, Side view of complex I showing surfaces for subunits B14.5a and B16.6. b, IMS view of complex I showing surfaces for subunits SGDH and PDSW. The point at which the two subunits are intertwined is marked with a star. c, View of the hydrophilic arm looking from above the membrane arm. The surface of the 18-kDa subunit that spans the hydrophilic arm is shown. d, Matrix view of the tip of the membrane arm with the surface of supernumerary subunit B22 shown. e, Close up of the centre of the membrane arm on the IMS side. This region contains many interactions between supernumerary subunits and the side chains of residues involved are shown. The region is also a hot spot for cross-links, the side chains involved are shown and cross-links are indicated with dashed lines (acid cross-links: red; basic cross-links: blue). f, Close up of the C-terminal helix of supernumerary subunit PDSW at the centre of the membrane arm on the IMS side. This helix extends away from complex I and is encircled by the C termini of supernumerary subunits B14.5b, ESSS and B15. The side chains of residues involved in stabilizing interactions are shown. A possible disulfide bond between PDSW (Cys154) and ESSS (Cys112) and stabilizing salt bridges are indicated by dashed lines. Subunits are coloured as in Fig. 1.

Extended Data Figure 8 Comparison of ‘open’ and ‘closed’ 3D class structures.

a, b, Side (a) and top (b) view from the matrix for the alignment of the open class structure (in cyan) and closed class structure (in grey). To generate the closed class structure, the final structure of the open class was refined in real space in Phenix (5 macro cycles with morphing at each cycle) against 4.6 Å map of the closed class (Extended Data Fig. 1). All of the α-helices were well fit into density, but owing to low resolution of the closed class no further refinement was performed and the comparison of structures involves only the relative positions of secondary structure elements. The two structures were aligned via transmembrane core subunits and are displayed as cartoon models. In the closed class the peripheral arm undergoes a hinge-like motion around the Q site towards the tip of the membrane arm, with the direction of shift indicated by the arrow in b. As a result, subunit B13 moves ~3 Å closer to the 42-kDa subunit, allowing for direct contacts. The shift is larger at the periphery, reaching 7 Å at the tip of the peripheral arm. Additionally, subunit ND5 and its matrix bulge move about 3 Å towards peripheral arm.

Extended Data Table 1 Summary of the model
Full size table
Extended Data Table 2 Summary of interactions between subunits of ovine complex I
Full size table

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Fiedorczuk, K., Letts, J., Degliesposti, G. et al. Atomic structure of the entire mammalian mitochondrial complex I. Nature 538, 406–410 (2016). https://doi.org/10.1038/nature19794

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