Architecture of mammalian respiratory complex I

Journal name:
Nature
Volume:
515,
Pages:
80–84
Date published:
DOI:
doi:10.1038/nature13686
Received
Accepted
Published online

Abstract

Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved ‘core’ subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 ‘supernumerary’ subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron–sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.

At a glance

Figures

  1. Overall map for complex I from B. taurus heart mitochondria determined by single-particle cryo-EM.
    Figure 1: Overall map for complex I from B. taurus heart mitochondria determined by single-particle cryo-EM.

    Three distinct features of the complex are revealed by overlaying maps at different density thresholds. The map at the highest threshold (red) reveals the FeS clusters. The map at medium threshold (grey) reveals the overall architecture of the protein and the 78 TMHs in the membrane domain. The detergent–phospholipid belt observed as a dominant feature at low density threshold (translucent blue) represents the density that remains around the membrane domain after cutting out the final model of the protein, and denotes the position of the complex in the membrane. It is ~30 Å thick, and 3–4 Å thinner at the proximal end of the complex (left) than at the distal end (right).

  2. Structures of the core subunits of mammalian complex I.
    Figure 2: Structures of the core subunits of mammalian complex I.

    a, Structural models of the 14 mammalian core subunits (cartoon representation) and their density (transparent surface); the subunits are coloured individually and labelled with text in the same colours. The chain of FeS clusters is shown modelled to the highest density peaks (blue mesh) in the inset. b, The seven membrane-bound mammalian core subunits, viewed from the matrix. Arrows indicate the positions of the four TMHs in T. thermophilus that are not present in B. taurus: three N-terminal TMHs in ND2 and one C-terminal TMH in ND1. The position of TMH4 in ND6 is different in B. taurus and T. thermophilus (marked with stars). For a detailed comparison of the B. taurus and T. thermophilus structures see Extended Data Figs 4 and 5.

  3. Architecture of mammalian complex I showing the densities of the supernumerary subunits enclosing the core domain.
    Figure 3: Architecture of mammalian complex I showing the densities of the supernumerary subunits enclosing the core domain.

    The models for the core subunits are in light colours (as labelled) in surface representation, and density attributed to the supernumerary subunits, forming a cage around the core subunits, is in dark red. The supernumerary subunits are concentrated on each side of the membrane domain, and around the lower section of the hydrophilic domain. The NADH-binding site in the 51 kDa subunit is indicated, with the predicted positions for the flavin isoalloxazine (orange spheres) and three conserved phenylalanines at the entry to the site (yellow); the vicinity of this site is devoid of supernumerary subunit density.

  4. Structural assignments of supernumerary subunits in mammalian complex I.
    Figure 4: Structural assignments of supernumerary subunits in mammalian complex I.

    a, A semi-transparent surface for the density map for mammalian complex I is shown in pale grey, with the surface from the core subunits in wheat. Structural models for the supernumerary subunits are shown in colour and labelled accordingly (dashed lines indicate subunits on the back of the structure). Subunits labelled with brackets are those with less certain assignments, and structural elements, which cannot be assigned confidently in the current map, are in blue. b, Arrangement of TMHs, viewed from the matrix. The core subunits are in light colours (wheat for ND1, ND4 and ND5, green for ND2, ND3, ND4L and ND6). The supernumerary subunits are coloured as in a.

  5. Structural models for supernumerary subunits in mammalian complex I.
    Figure 5: Structural models for supernumerary subunits in mammalian complex I.

    a, Models for three supernumerary subunits in cartoon representation, coloured from blue to red (N to C termini). b, Structural models and relationships of supernumerary subunits to the core structure. B14.7 is located at the end of the transverse helix, next to ND5–TMH16. The density assigned to PGIV forms an L-shaped ‘clip’ over B16.6, which bends around the heel at ND1. Finally, the supernumerary subunits around the lower section of the hydrophilic domain are viewed in cartoon representation from the matrix. The core membrane subunits (white) and four core hydrophilic subunits, the 49 kDa (blue), 30 kDa (green), PSST (yellow) and TYKY (cyan) subunits, are shown in surface representation.

  6. Single-particle cryo-EM analysis of B. taurus complex I.
    Extended Data Fig. 1: Single-particle cryo-EM analysis of B. taurus complex I.

    a, Typical micrograph of complex I particles imaged after freezing in vitreous ice on a holey-carbon grid. Some of the selected particles are marked with red boxes. Scale bar, 50 nm. b, Two-dimensional reference classification showing particles lying in different orientations in the ice. The size of each box is 280 pixels and the two-dimensional classification was made in RELION14.

  7. Validation of the map and resolution.
    Extended Data Fig. 2: Validation of the map and resolution.

    a, Tilt-pair analysis45 of complex I in Cymal-7. One-hundred complex I particles from eight image pairs, recorded with a relative tilt angle of 10°, were extracted and subjected to tilt-pair analysis with FREALIGN42. The outer radius of the plot is 40° and the orange circle centred at the expected tilt angle has a radius of 6°. b, Phase randomization to check for overfitting. Phases that are beyond 10 Å in each of the micrographs used in the final data set (frames 1–32) were randomized, and then refinement was performed as for a normal data set (FSC summed image corresponding to frames 1–32). As expected, the graph shows a drop in the Fourier shell correlation (FSC) curve at 10 Å, validating the presence of information beyond 10 Å in the images. Note that the use of gold-standard refinement procedures in RELION14 prevents any overfitting, and this test was done only as an additional control. c, An overview of the final map and the model built into it. d, FSC curves of the final map and of the model versus the map. The curve in red is the gold-standard FSC of the final map (after classification) and the resolution at FSC = 0.143 is ~4.95 Å. The curve in cyan is the FSC between the final map and the model, and at FSC = 0.5 the resolution is 6.7 Å. Note that the present model is not complete since it is only a polyalanine model without any side chains, and loop regions in a number of subunits have not been modelled. e, The final map of mammalian complex I was analysed with ResMap49. The left-hand panel (with lower density threshold) shows that the detergent–phospholipid belt is of lower resolution, and most of the protein regions of the map show resolution distributed from 5 to 6 Å. In the right-hand panel the map is shown at a higher density threshold, so the detergent–phospholipid belt is not visualized. Some of the interior parts of the map have resolution of 4.8–5 Å.

  8. Example regions of the density map with the model fitted to the map.
    Extended Data Fig. 3: Example regions of the density map with the model fitted to the map.

    a, ND2 is shown from the membrane plane, highlighting the densities for three aromatic side chains and one of the helix-breaking loops. b, Subunit ND4 viewed from the matrix. c, The density for a [4Fe–4S] cluster and surrounding protein is shown in the PSST subunit. d, A region of the 49 kDa subunit shows a well resolved α-helical stretch and aromatic side chains, and the β-strands are beginning to be resolved. e, Subunit B8 is an example of a supernumerary subunit in a peripheral region of the molecule. f, Density consistent with a bound nucleotide is observed in the 39 kDa subunit, in a similar position to in homologous structures and as expected from analysis of Y. lipolytica complex I (ref. 27). However, the present resolution of the map precludes the inclusion of this nucleotide in the final model.

  9. Global comparison of the core subunit structures of bacterial and mammalian complex I.
    Extended Data Fig. 4: Global comparison of the core subunit structures of bacterial and mammalian complex I.

    The core subunits from B. taurus are in blue, and from T. thermophilus (PDB accession 4HEA4) in orange. The structures have been superimposed using ND1 (the heel subunit). Top: the ND2, ND4 and ND5 domain is rotated in B. taurus relative to in T. thermophilus, increasing the curvature in the B. taurus membrane domain. The complex is viewed along the 11° rotation vector (orange) that maps the T. thermophilus ND2, ND4 and ND5 domain to the B. taurus domain, along with a small 5 Å translation to superimpose the domain centres. Correspondingly, the ND3, ND4L and ND6 domains are superimposed by a 4° rotation and a 1 Å translation. Rotation of ND2, 4 and 5 about the long axis of the domain, as noted for Y. lipolytica58, is not observed. Bottom: the NADH dehydrogenase domain containing the 51 and 24 kDa subunits is rotated by 23° and translated by 14 Å in B. taurus, relative to in T. thermophilus, causing the FeS chains to diverge as the distance from ND1 increases. A similar rotation was observed in Y. lipolytica58. The complex is viewed from behind ND1. Correspondingly, the 49 kDa, PSST and TYKY subunits are superimposed by a 6° rotation and a 2 Å translation. The structures were analysed using Superpose from the CCP4 suite59 and the 75 kDa and 30 kDa subunits were not included due to their lower structural conservation.

  10. Comparison of the individual structures of the core subunits of bacterial and mammalian complex I.
    Extended Data Fig. 5: Comparison of the individual structures of the core subunits of bacterial and mammalian complex I.

    a, The structure of each subunit from T. thermophilus (wheat) (PDB accession 4HEA4) has been superimposed separately on its corresponding subunit from B. taurus (coloured as labelled) with the transverse helix plus TMH16 of ND5 also aligned separately. The complexes are viewed from behind ND1 (top), from the side (middle) and from the matrix (bottom, ND subunits only). b, Observed differences in the structures of the core subunits of B. taurus and T. thermophilus complexes I. Grey, conserved structure from B. taurus and T. thermophilus (PDB accession 4HEA4); red, structural elements present only in T. thermophilus; blue, structural elements present only in B. taurus. The C-terminal domain of the 75 kDa subunit is not resolved in B. taurus, but its structure is clearly different to in T. thermophilus.

Tables

  1. Reference table for the nomenclature of the core subunits of complex I
    Extended Data Table 1: Reference table for the nomenclature of the core subunits of complex I
  2. Summary of the models of the core subunits of B. taurus complex I
    Extended Data Table 2: Summary of the models of the core subunits of B. taurus complex I
  3. Distances between the redox cofactors in structural models of complex I
    Extended Data Table 3: Distances between the redox cofactors in structural models of complex I
  4. Knowledge about the supernumerary subunits of B. taurus complex I
    Extended Data Table 4: Knowledge about the supernumerary subunits of B. taurus complex I
  5. Summary of the models of the supernumerary subunits of B. taurus complex I
    Extended Data Table 5: Summary of the models of the supernumerary subunits of B. taurus complex I

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Hirst, J. Mitochondrial complex I. Annu. Rev. Biochem. 82, 551575 (2013)
  2. Carroll, J., Fearnley, I. M., Shannon, R. J., Hirst, J. & Walker, J. E. Analysis of the subunit composition of complex I from bovine heart mitochondria. Mol. Cell. Proteomics 2, 117126 (2003)
  3. Hirst, J., Carroll, J., Fearnley, I. M., Shannon, R. J. & Walker, J. E. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604, 135150 (2003)
  4. Baradaran, R., Berrisford, J. M., Minhas, G. S. & Sazanov, L. A. Crystal structure of the entire respiratory complex I. Nature 494, 443448 (2013)
  5. Efremov, R. G. & Sazanov, L. A. Structure of the membrane domain of respiratory complex I. Nature 476, 414420 (2011)
  6. Efremov, R. G., Baradaran, R. & Sazanov, L. A. The architecture of respiratory complex I. Nature 465, 441445 (2010)
  7. Sazanov, L. A. & Hinchliffe, P. Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 311, 14301436 (2006)
  8. Hunte, C., Zickermann, V. & Brandt, U. Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 329, 448451 (2010)
  9. Leonard, K., Haiker, H. & Weiss, H. Three-dimensional structure of NADH:ubiquinone reductase (complex I) from Neurospora mitochondria determined by electron microscopy of membrane crystals. J. Mol. Biol. 194, 277286 (1987)
  10. Grigorieff, N. Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 Å in ice. J. Mol. Biol. 277, 10331046 (1998)
  11. Clason, T. et al. The structure of eukaryotic and prokaryotic complex I. J. Struct. Biol. 169, 8188 (2010)
  12. Fassone, E. & Rahman, S. Complex I deficiency: clinical features, biochemistry and molecular genetics. J. Med. Genet. 49, 578590 (2012)
  13. Sharpley, M. S., Shannon, R. J., Draghi, F. & Hirst, J. Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria. Biochemistry 45, 241248 (2006)
  14. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519530 (2012)
  15. Bai, X.-C., Fernandez, I. S., McMullan, G. & Scheres, S. H. W. Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461 (2013)
  16. Birrell, J. A. & Hirst, J. Truncation of subunit ND2 disrupts the threefold symmetry of the antiporter-like subunits in complex I from higher metazoans. FEBS Lett. 584, 42474252 (2010)
  17. Kussmaul, L. & Hirst, J. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc. Natl Acad. Sci. USA 103, 76077612 (2006)
  18. Mimaki, M., Wang, X., McKenzie, M., Thorburn, D. R. & Ryan, M. T. Understanding mitochondrial complex I assembly in health and disease. Biochim. Biophys. Acta 1817, 851862 (2012)
  19. Dieteren, C. E. J. et al. Subunit-specific incorporation efficiency and kinetics in mitochondrial complex I homeostasis. J. Biol. Chem. 287, 4185141860 (2012)
  20. Brockmann, C. et al. The oxidised subunit B8 from human complex I adopts a thioredoxin fold. Structure 12, 16451654 (2004)
  21. Keeney, P. M., Xie, J., Capaldi, R. A. & Bennett, J. P. Parkinson’s disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J. Neurosci. 26, 52565264 (2006)
  22. Leshinsky-Silver, E. et al. NDUFS4 mutations cause Leigh syndrome with predominant brainstem involvement. Mol. Genet. Metab. 97, 185189 (2009)
  23. Kirby, D. M. et al. NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency. J. Clin. Invest. 114, 837845 (2004)
  24. Sharpley, M. S. Studies of the Catalytic Activity of NADH:Ubiquinone Oxidoreductase (Complex I) from Bovine Mitochondria. PhD thesis, Cambridge Univ. (2005)
  25. Morais, V. A. et al. PINK1 loss of function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 344, 203207 (2014)
  26. Fearnley, I. M. & Walker, J. E. Conservation of sequences of subunits of mitochondiral complex I and their relationships with other proteins. Biochim. Biophys. Acta 1140, 105134 (1992)
  27. Abdrakhmanova, A., Zwicker, K., Kerscher, S., Zickermann, V. & Brandt, U. Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex. Biochim. Biophys. Acta 1757, 16761682 (2006)
  28. Babot, M. et al. ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I. Biochim. Biophys. Acta 1837, 929939 (2014)
  29. Runswick, M. J., Fearnley, I. M., Skehel, J. M. & Walker, J. E. Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria. FEBS Lett. 286, 121124 (1991)
  30. Cronan, J. E., Fearnley, I. M. & Walker, J. E. Mammalian mitochondria contain a soluble acyl carrier protein. FEBS Lett. 579, 48924896 (2005)
  31. Dobrynin, K. et al. Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica. Biochim. Biophys. Acta 1797, 152159 (2010)
  32. Angerer, H. et al. The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity. Proc. Natl Acad. Sci. USA 111, 52075212 (2014)
  33. Andrews, B., Carroll, J., Ding, S., Fearnley, I. M. & Walker, J. E. Assembly factors for the membrane arm of human complex I. Proc. Natl Acad. Sci. USA 110, 1893418939 (2013)
  34. Angerer, H. et al. A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I. Biochem. J. 437, 279288 (2011)
  35. Fearnley, I. M. et al. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I). J. Biol. Chem. 276, 3834538348 (2001)
  36. Banci, L. et al. Structural characterization of CHCHD5 and CHCHD7: two atypical human twin CX9C proteins. J. Struct. Biol. 180, 190200 (2012)
  37. Szklarczyk, R. et al. NDUFB7 and NDUFA8 are located at the intermembrane surface of complex I. FEBS Lett. 585, 737743 (2011)
  38. Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107112 (2013)
  39. Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 14851489 (2014)
  40. Allegretti, M., Mills, D. J., McMullan, G., Kühlbrandt, W. & Vonck, J. Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector. eLife 3, e01963 (2014)
  41. Bellare, J. R., Davis, H. T., Scriven, L. E. & Talmon, Y. Controlled environment vitrification system: an improved sample preparation technique. J. Electron Microsc. Tech. 10, 87111 (1988)
  42. Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117125 (2007)
  43. Smith, J. M. XIMDISP—a visualization tool to aid structure determination from electron microscope images. J. Struct. Biol. 125, 223228 (1999)
  44. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 3846 (2007)
  45. Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, 10281046 (2011)
  46. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334347 (2003)
  47. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 2435 (2013)
  48. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721745 (2003)
  49. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 6365 (2014)
  50. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486501 (2010)
  51. Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195202 (1999)
  52. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567580 (2001)
  53. Tusnády, G. E. & Simon, I. Principles governing amino acid composition of integral membrane proteins: applications to topology prediction. J. Mol. Biol. 283, 489506 (1998)
  54. Bernsel, A., Viklund, H., Hennerdal, A. & Elofsson, A. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res. 37, W465W468 (2009)
  55. Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244W248 (2005)
  56. Eswar, N. et al. Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinform. Chapter 5, Unit 5.6. (2006)
  57. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195201 (2006)
  58. Efremov, R. G. & Sazanov, L. A. Respiratory complex I: ‘steam engine’ of the cell? Curr. Opin. Struct. Biol. 21, 532540 (2011)
  59. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 22562268 (2004)
  60. Balsa, E. et al. NDUFA4 is a subunit of complex IV of the mammalian electron transport chain. Cell Metab. 16, 378386 (2012)
  61. Johansson, K. et al. Structural basis for substrate specificities of cellular deoxyribonucleoside kinases. Nature Struct. Biol. 8, 616620 (2001).
  62. King, J. D. et al. Predicting protein function from structure - the roles of short-chain dehydrogenase/reductase enzymes in Bordetella O-antigen biosynthesis. J. Mol. Biol. 374, 749763 (2007)
  63. Parris, K. D. et al. Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites. Structure 8, 883895 (2000)

Download references

Author information

  1. These authors contributed equally to this work.

    • Kutti R. Vinothkumar &
    • Jiapeng Zhu

Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Kutti R. Vinothkumar
  2. MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK

    • Jiapeng Zhu &
    • Judy Hirst

Contributions

K.R.V. carried out EM experiments and analysis; J.Z. prepared protein; K.R.V., J.Z. and J.H. modelled and analysed data; J.H. designed the project; K.R.V., J.Z. and J.H. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

The EM map of complex I has been deposited in the Electron Microscopy Data Bank under accession number EMD-2676, and the associated model has been deposited in the Protein Data Bank under accession number 4UQ8.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Single-particle cryo-EM analysis of B. taurus complex I. (672 KB)

    a, Typical micrograph of complex I particles imaged after freezing in vitreous ice on a holey-carbon grid. Some of the selected particles are marked with red boxes. Scale bar, 50 nm. b, Two-dimensional reference classification showing particles lying in different orientations in the ice. The size of each box is 280 pixels and the two-dimensional classification was made in RELION14.

  2. Extended Data Figure 2: Validation of the map and resolution. (593 KB)

    a, Tilt-pair analysis45 of complex I in Cymal-7. One-hundred complex I particles from eight image pairs, recorded with a relative tilt angle of 10°, were extracted and subjected to tilt-pair analysis with FREALIGN42. The outer radius of the plot is 40° and the orange circle centred at the expected tilt angle has a radius of 6°. b, Phase randomization to check for overfitting. Phases that are beyond 10 Å in each of the micrographs used in the final data set (frames 1–32) were randomized, and then refinement was performed as for a normal data set (FSC summed image corresponding to frames 1–32). As expected, the graph shows a drop in the Fourier shell correlation (FSC) curve at 10 Å, validating the presence of information beyond 10 Å in the images. Note that the use of gold-standard refinement procedures in RELION14 prevents any overfitting, and this test was done only as an additional control. c, An overview of the final map and the model built into it. d, FSC curves of the final map and of the model versus the map. The curve in red is the gold-standard FSC of the final map (after classification) and the resolution at FSC = 0.143 is ~4.95 Å. The curve in cyan is the FSC between the final map and the model, and at FSC = 0.5 the resolution is 6.7 Å. Note that the present model is not complete since it is only a polyalanine model without any side chains, and loop regions in a number of subunits have not been modelled. e, The final map of mammalian complex I was analysed with ResMap49. The left-hand panel (with lower density threshold) shows that the detergent–phospholipid belt is of lower resolution, and most of the protein regions of the map show resolution distributed from 5 to 6 Å. In the right-hand panel the map is shown at a higher density threshold, so the detergent–phospholipid belt is not visualized. Some of the interior parts of the map have resolution of 4.8–5 Å.

  3. Extended Data Figure 3: Example regions of the density map with the model fitted to the map. (974 KB)

    a, ND2 is shown from the membrane plane, highlighting the densities for three aromatic side chains and one of the helix-breaking loops. b, Subunit ND4 viewed from the matrix. c, The density for a [4Fe–4S] cluster and surrounding protein is shown in the PSST subunit. d, A region of the 49 kDa subunit shows a well resolved α-helical stretch and aromatic side chains, and the β-strands are beginning to be resolved. e, Subunit B8 is an example of a supernumerary subunit in a peripheral region of the molecule. f, Density consistent with a bound nucleotide is observed in the 39 kDa subunit, in a similar position to in homologous structures and as expected from analysis of Y. lipolytica complex I (ref. 27). However, the present resolution of the map precludes the inclusion of this nucleotide in the final model.

  4. Extended Data Figure 4: Global comparison of the core subunit structures of bacterial and mammalian complex I. (1,096 KB)

    The core subunits from B. taurus are in blue, and from T. thermophilus (PDB accession 4HEA4) in orange. The structures have been superimposed using ND1 (the heel subunit). Top: the ND2, ND4 and ND5 domain is rotated in B. taurus relative to in T. thermophilus, increasing the curvature in the B. taurus membrane domain. The complex is viewed along the 11° rotation vector (orange) that maps the T. thermophilus ND2, ND4 and ND5 domain to the B. taurus domain, along with a small 5 Å translation to superimpose the domain centres. Correspondingly, the ND3, ND4L and ND6 domains are superimposed by a 4° rotation and a 1 Å translation. Rotation of ND2, 4 and 5 about the long axis of the domain, as noted for Y. lipolytica58, is not observed. Bottom: the NADH dehydrogenase domain containing the 51 and 24 kDa subunits is rotated by 23° and translated by 14 Å in B. taurus, relative to in T. thermophilus, causing the FeS chains to diverge as the distance from ND1 increases. A similar rotation was observed in Y. lipolytica58. The complex is viewed from behind ND1. Correspondingly, the 49 kDa, PSST and TYKY subunits are superimposed by a 6° rotation and a 2 Å translation. The structures were analysed using Superpose from the CCP4 suite59 and the 75 kDa and 30 kDa subunits were not included due to their lower structural conservation.

  5. Extended Data Figure 5: Comparison of the individual structures of the core subunits of bacterial and mammalian complex I. (508 KB)

    a, The structure of each subunit from T. thermophilus (wheat) (PDB accession 4HEA4) has been superimposed separately on its corresponding subunit from B. taurus (coloured as labelled) with the transverse helix plus TMH16 of ND5 also aligned separately. The complexes are viewed from behind ND1 (top), from the side (middle) and from the matrix (bottom, ND subunits only). b, Observed differences in the structures of the core subunits of B. taurus and T. thermophilus complexes I. Grey, conserved structure from B. taurus and T. thermophilus (PDB accession 4HEA4); red, structural elements present only in T. thermophilus; blue, structural elements present only in B. taurus. The C-terminal domain of the 75 kDa subunit is not resolved in B. taurus, but its structure is clearly different to in T. thermophilus.

Extended Data Tables

  1. Extended Data Table 1: Reference table for the nomenclature of the core subunits of complex I (144 KB)
  2. Extended Data Table 2: Summary of the models of the core subunits of B. taurus complex I (290 KB)
  3. Extended Data Table 3: Distances between the redox cofactors in structural models of complex I (165 KB)
  4. Extended Data Table 4: Knowledge about the supernumerary subunits of B. taurus complex I (275 KB)
  5. Extended Data Table 5: Summary of the models of the supernumerary subunits of B. taurus complex I (201 KB)

Additional data