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.
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Accession codes
Primary accessions
Electron Microscopy Data Bank
Protein Data Bank
Referenced accessions
Protein Data Bank
Data deposits
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.
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Acknowledgements
We thank R. Henderson, S. H. W. Scheres, G. McMullan, G. Murshudov, P. Emsley and J. E. Walker for helpful advice, the FEI fellows for educating us on use of the Titan Krios, J. Grimmett and T. Darling for computational help, and S. Chen and C. Savva for EM help. This work was supported by the Medical Research Council, grant numbers U105184322 (K.R.V., in R. Henderson’s group) and U105663141 (J.H.).
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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.
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Extended data figures and tables
Extended Data Figure 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.
Extended Data Figure 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.
Extended Data Figure 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.
Extended Data Figure 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.
Extended Data Figure 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.
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Vinothkumar, K., Zhu, J. & Hirst, J. Architecture of mammalian respiratory complex I. Nature 515, 80–84 (2014). https://doi.org/10.1038/nature13686
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DOI: https://doi.org/10.1038/nature13686
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