The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4 Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.
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Protein Data Bank
The 3D cryo-EM density map has been deposited in the Electron Microscopy Data Bank (EMDB), with accession code EMD- 9534. The coordinates of atomic models have been deposited in the Protein Data Bank (PDB) under the accession code 5GPN for the respirasome.
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We would like to thank the staff members X. Li, F. Yang and Y. Li of Tsinghua University Branch of China National Center for Protein Sciences (Beijing, China) for providing the facility support. This work was supported by funds from the Ministry of Science and Technology (2016YFA0501101 and 2012CB911101 to M.J., and 2013CB910404 and 2016YFA0500700 to N.G.), and the National Outstanding Young Scholar Science Foundation and National Natural Science Foundation of China (31030020 and 31170679 to M.Y., 31422016 to N.G.).
The authors declare no competing financial interests.
Nature thanks A. Engel, D. Winge, and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Fractions of sucrose gradient ultracentrifugation were analysed by BNPA. b, In-gel staining of the native gel by NBT. Higher-molecular-weight bands were indicated by a red arrow. c, A representative trace of size-exclusion chromatography of the respiratory supercomplexes. d, Protein samples of the size-exclusion chromatography fractions were subjected to BNPA. Fractions of 9–9.5 ml (elution volume) were used for negative staining EM and cryo-EM. e, NBT staining of the size-exclusion chromatography BNPA.
a, A representative micrograph of negatively stained respirasomes. b, 2D class averages of negatively stained particles. c, A flowchart of th einitial model generation and validation. A density ball was used as the initial reference for 3D classification. Results of 3D classification are shown on the right, with class 1, class 2 and class 3 containing 3,031, 2,308 and 4,597 particles, respectively. Bottom left shows the final model used for the 3D refinement of cryo-EM particles.
a, A representative cryo-EM micrograph of respirasome. b, Power-spectrum of the micrograph in a. The white circle indicates the 3.0 Å frequency. c, A collection of raw particles of the respirasome collected with Titan Krios (300 kV) and Falcon II. d, Representative 2D class averages in different views.
Workflow of 3D reconstruction with cryo-EM data. A total of 81,100 particles were kept after 2D classification, and subject to two rounds of 3D classification. A final data set containing ~50,000 particles were used for high-resolution refinement (see Methods for more details).
a, Local resolution map of the final 3D density map. From left to right are respectively side, top and bottom views. b, Gold-standard Fourier shell correlation (FSC) curve of the final density map, after correction of the soft-mask-induced effects. c, Particle orientation distributions in the last iteration of the structural refinement. Red cylinders mean more particles on these orientations. Heights of cylinders represent the relative numbers of particles.
a, Gold-standard fourier shell Correlation (FSC) curves of the density maps of CI and CIII obtained by subregion refinement. Both the maps of CI and CIII were refined to a resolution of 3.97 Å. b, The density map (blue meshes) of CI is displayed at root mean squared deviation (r.m.s.d.) = 12 contour level. The backbones of core subunits are shown in the density. c, The density map (blue meshes) of the TM helices of ND2 is displayed at r.m.s.d. = 12 contour level to illustrate the well-resolved side chains of ND2 at a resolution of 3.97 Å after subregion refinement. The residues are shown in line representation and three of them are labelled. The figure was prepared with Coot. d, CIII in our structure of the respirasome is in the intermediate state. The distances between [2Fe-2S] to haem c1, and [2Fe-2S] to haem bL in the final refined CIII structure are 30 Å and 27 Å, respectively. Different subunits are coloured individually as indicated. The figure was generated using PyMOL.
The expected positions of cytochrome c are based on the fitting of the structures of CIII with bound cytochrome c from yeast (PDB accession code 3CX5)67. The two cytochrome c molecules of CIII are shown as blue and pink cartoons, respectively. The transmembrane region is indicated by two dashed lines. M, matrix; IM, inner membrane; IMS, intermembrane space.
a, The density map (blue meshes) of NDUFA11 is displayed at r.m.s.d. = 12 contour level. The backbone is shown in line and the N and C termini are indicated. b, The density map (blue meshes) of NDUFAB1 and NDUFB9 is displayed at r.m.s.d. = 12 contour level. The backbones are coloured in orange and yellow, respectively, and the N and C termini are indicated.
. a. Sequence alignment of the NDUFB9 subunit of CI from different species. b, Sequence alignment of the NDUFA11 subunit of CI from different species. c, Sequence alignment of the UQCRC1 binding motif from different species. All alignments were carried out using DNAMAN.
a, Conformational changes of the core and assigned supernumerary subunits of CI. The model of CI in the respirasome (with subunits individually coloured) is globally aligned to the model of free CI (PDB accession code 4UQ8) (coloured in cyan). b, Comparison of the seven matrix core subunits of CI in the free form and in the respirasome. The polypeptides are indicated in different colours and labelled with text in the same colour. c, Same as b, but indicating the comparison of the seven membrane-bound core subunits. The bottom panel is viewed from the matrix side.
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Gu, J., Wu, M., Guo, R. et al. The architecture of the mammalian respirasome. Nature 537, 639–643 (2016). https://doi.org/10.1038/nature19359
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