Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.
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Huntingtin structure is orchestrated by HAP40 and shows a polyglutamine expansion-specific interaction with exon 1
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Protein Data Bank
The cryo-EM map of the 39S mitoribosomal subunit has been deposited in the Electron Microscopy Databank with accession code EMD-2490. The coordinates of the cryo-EM-based model of the 39S mitoribosomal subunit have been deposited in the Protein Data Bank under accession code 4CE4.
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Cryo-EM data were collected at the electron microscopy facility of ETH Zurich (EMEZ) and at FEI Company Eindhoven. We thank P. Tittmann, F. de Haas and K. Sader for support. We acknowledge the use of computing infrastructure provided by the Central Information Technology Services of ETH Zurich. This work was supported by the Swiss National Science Foundation (SNSF), the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF, European Research Council (ERC) grant 250071 under the European Community’s Seventh Framework Programme (to N.B.), the Commission of the European Communities through the PROSPECTS consortium (EU FP7 projects 201648, 233226) (R.A.) and the European Research Council (ERC-2008-AdG 233226) (R.A.).
The authors declare no competing financial interests.
Extended data figures and tables
a, Fourier Shell Correlation (FSC) curve of the cryo-EM reconstruction of the 39S mitoribosomal subunit. The resolution estimate is 4.9 Å according to the FSC = 0.143 criterion (gold standard FSC)38. b, Multiparticle refinement of 39S subunit data sets obtained using the FEI Falcon I and Falcon II direct electron detectors. Particle numbers in the classes are indicated below the volumes. Class 1 was used for further refinement of the structure of the 39S subunit to higher resolution.
a, b, View from the solvent side (a) and from the subunit interface side (b) of the 39S mitoribosomal subunit cryo-EM map, segmented into ribosomal proteins (gold) and rRNA (light blue). CP, central protuberance; L1, L1 stalk.
Deletions of rRNA secondary structure elements in mitochondrial 16S rRNA are indicated in red on the secondary structure diagram of the Escherichia coli 23S rRNA. Segments for which the precise locations of deletions cannot be deduced from our cryo-EM map (the L1 stalk, a part of domain I rRNA, and the connection to the L7/L12 stalk) are indicated in grey. Depiction is based on the secondary structure of the bacterial 23S rRNA53 (template obtained from the Noller laboratory web page http://rna.ucsc.edu/rnacenter/noller_lab.html).
Extended Data Figure 4 Secondary structure diagram of the S. scrofa large mitoribosomal subunit rRNA.
Domains are coloured according to Fig. 2 and labelled by Roman numerals. Nucleotides not built in our structure are printed in grey. Bacterial secondary structure elements missing in mammalian mitoribosomes are schematically indicated in red. Watson–Crick base pairs are indicated by lines (−), G•U base pairs by dots, and nonstandard base pairs by rings. Depiction is based on the secondary structure of bacterial 23S rRNA53 (template obtained from the Noller lab web page http://rna.ucsc.edu/rnacenter/noller_lab.html).
a, Depiction of the mitoribosomal central protuberance RNA (dark blue) and the neighbouring protein MRPL18 (cyan). b, Depiction of the bacterial 5S rRNA (orange) in complex with ribosomal protein L18 (khaki; PDB ID 3V2D). c, Overlay of a and b according to the homologous proteins L18 and MRPL18. d, Overview of the 39S subunit with the fitted RNAs for orientation. e, In bacterial ribosomes, the 5S rRNA connects the central protuberance to the main body of the subunit (the contact region is indicated by an arrow). However, no density for this connection can be seen in the cryo-EM map of the 39S subunit (grey). f, In the mitochondrial 39S subunit, a long α-helical protein density corresponding to MRPL52 (gold) instead of the 5S rRNA connects the mitoribosomal central protuberance (MRPL38, purple) to the subunit body.
Extended Data Figure 6 Quality of the fits of newly positioned 39S mitoribosomal proteins in the cryo-EM density.
Depiction of the cryo-EM density (first column), fits of the unchanged homology models (second column), and fits of the adjusted final models (third column) of MRPL45 (a–c), MRPL44 (d–f), MRPL39 (g–i), MRPL38 (j–l), ICT1 (m–o) and MRPL49 (p–r). The 39S cryo-EM map is shown at two threshold levels (red and grey, respectively).
The secondary structure prediction (output from Phyre2)19 shows a very long (approximately 50 amino acid) α-helix. Blue stars indicate crosslinks of the modelled domain of MRPL38 to MRPL52, as observed in CX-MS experiments.
a, b, Comparison of the conserved ring of proteins around the polypeptide tunnel exit (indicated by an asterisk). a, The polypeptide tunnel exit in bacteria17 (PDB ID 3V2D). b, The polypeptide tunnel exit in the porcine mitoribosome. The ring of proteins around the tunnel exit is conserved. c, Additional mitoribosome-specific proteins and protein extensions are also located close to the tunnel exit. The newly identified MRPL39, MRPL44 and MRPL45 are shown in green, red and blue, respectively.
Extended Data Figure 9 The binding sites of the newly docked mitoribosome-specific proteins MRPL44 and MRPL45.
Crosslinks are indicated by double arrows (see also Supplementary Table 3) unless both crosslinking residues have been modelled in the structure. a, MRPL44 (dark red) has been crosslinked to MRPL20 (gold). The crosslinked sites on MRPL44 and MRPL20 are indicated by green and blue spheres, respectively. b, MRPL45 (dark blue) has been crosslinked to MRPL24 (gold) and MRPL23 (orange) in S. scrofa, as well as MRPL22 (pink) and MRPL39 (green) in B. taurus. For all of these crosslinks, at least one crosslink site is located on mitochondria-specific extensions that could not be modelled, and therefore these crosslinks cannot be mapped precisely in the structure. c, Mutation in the human homologue of MRPL44 in mitochondrial infantile cardiomyopathy25. The L156R mutation (green) maps to a residue not involved in 39S binding. The affected residue is located between two α-helices of the RNase domain, and its mutation probably perturbs the structure of MRPL44 and its capacity to bind the 39S subunit.
a, MRPL45 (blue) may anchor the 39S subunit (grey) to the mitochondrial inner membrane. The membrane surface is tentatively indicated by a horizontal line. b, The structure of the C-terminal domain of TIM44 (ref. 54) (PDB ID 2FXT) with its putative membrane-interacting segment30 is indicated in red. c, The part of MRPL45 corresponding to the putative membrane anchoring segment of TIM44 is shown in red.
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Greber, B., Boehringer, D., Leitner, A. et al. Architecture of the large subunit of the mammalian mitochondrial ribosome. Nature 505, 515–519 (2014). https://doi.org/10.1038/nature12890
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