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The complete structure of the large subunit of the mammalian mitochondrial ribosome

Abstract

Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria1. Mammalian mitoribosomes, which comprise 39S and 28S subunits2, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes3,4,5. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit6, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.

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Figure 1: Overall structure of the 39S subunit.
Figure 2: The peptidyl transferase centre of the 39S subunit.
Figure 3: The mitoribosomal polypeptide exit tunnel.
Figure 4: The CP tRNA in the 39S subunit.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The 3.4 Å cryo-EM map of the 39S mitoribosomal subunit has been deposited in the Electron Microscopy Databank with accession code EMD-2787. The coordinates of the atomic structure of the 39S mitoribosomal subunit have been deposited in the Protein Data Bank under accession codes 4v1a and 4v19.

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Acknowledgements

Cryo-EM data were collected at the electron microscopy facility of ETH Zurich (ScopeM). We thank P. Tittmann (ScopeM) for support. We thank J. Yu and C. Ciaudo for discussions. This work was supported by the Swiss National Science Foundation (SNSF), the National Center of Excellence in Research (NCCR) Structural Biology program of the Swiss National Science Foundation (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.).

Author information

Authors and Affiliations

Authors

Contributions

P.B. and B.J.G. performed preparation of the mitoribosomes. B.J.G., P.B. and D.B. prepared cryo-EM samples. D.B. and B.J.G. acquired the cryo-EM data. B.J.G. and P.B. calculated the cryo-EM reconstructions with support from D.B. M.L. and B.J.G. built the atomic model. M.L, N.S. and D.B. performed coordinate refinement of the atomic model. M.L., B.J.G., P.B., D.B. and N.B. interpreted the structure. A.L. performed CX-MS experiments in the laboratory of R.A. All authors contributed to the final version of the paper.

Corresponding author

Correspondence to Nenad Ban.

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Competing interests

CFI–ETH Zürich has filed a patent application to use the coordinates of the 39S mitoribosomal subunit for development of compounds that can (1) specifically bind to the mitochondrial ribosome and interfere with mitochondrial translation or (2) specifically inhibit translation of pathogenic organisms without interfering with mitochondrial translation.

Extended data figures and tables

Extended Data Figure 1 Computational sorting of the 55S mitoribosome data sets.

a, To obtain a particle sub-population suitable for high-resolution structure determination, two data sets were computationally sorted in RELION9. 2D and 3D classifications (only representative classes are shown) of a data set of a total of roughly 560,000 particle images resulted in the selection of 141,700 55S mitoribosomal particles. The 55S particles were refined to obtain a reconstruction of the 55S mitoribosome (3.6 Å resolution). b, The map obtained for the mammalian 55S mitoribosome (28S subunit in pale yellow; 39S subunit in light blue) filtered to 6 Å resolution. Two tRNA molecules are seen in the intersubunit space (A-site tRNA in yellow; P-site tRNA in purple). c, Local resolution plot for the 55S reconstruction (surface of the map on the left, viewed in section at the right). The local resolution is clearly below the average of 3.6 Å in some parts of the map, particularly in the 28S subunit (4.1 Å on average for the 28S subunit). Refinement with a mask around the 39S subunit (see a and Extended Data Fig. 2) resulted in an improved map suitable for de novo model building in the 39S section of the map (3.4 Å resolution).

Extended Data Figure 2 The cryo-EM map of the 39S subunit within the 55S mitoribosome.

a, Surface rendering of the 39S subunit cryo-EM map at 3.4 Å resolution, viewed from the solvent side. Landmarks are indicated: CP, central protuberance; L1, L1 stalk; L7/L12, L7/L12 stalk base; TE, polypeptide tunnel exit region. b, Black: FSC curve of the 39S cryo-EM reconstruction. The resolution is 3.4 Å according to the FSC = 0.143 criterion53. Curve computed using RELION9 from auto-masked maps computed from data half-sets. Red: the FSC curve computed between the final reconstruction and the refined coordinates (see Extended Data Fig. 3) indicates a resolution of 3.4 Å according to the FSC = 0.5 criterion, which has to be used in this case53. c, Local resolution plot. dg, Examples for the quality of the density: an rRNA double helix (d), a base pair (e), interactions of protein and rRNA with many large side chains visible (f), and a Mg2+ ion coordinated by the rRNA (g).

Extended Data Figure 3 Coordinate refinement of the 39S subunit atomic model.

a, Model refinement and validation statistics. b, Distribution of B-factors in the atomic model of the 39S subunit. Higher B-factors at the periphery, particularly in the L7/L12 stalk and CP regions, reflect the lower local resolution in these areas (Extended Data Fig. 2c). c, Refinement weight optimization. FSC curves of the atomic coordinates refined into maps calculated from one-half of the data (red, FSC model versus map computed from the full data set; blue, FSC model versus half-set 1 (used for test refinement); green, model versus half-set 2 (not used for test refinement)) using a weight of 1.0 for the reciprocal space data (see Methods for details). d, As in c, but with a weight of 3.0. e, As in c, but with a weight of 0.5.

Extended Data Figure 4 Overview of protein folds in the 39S subunit.

Overview of the structures of the 39S subunit proteins. Cyan, homologues of bacterial ribosomal proteins, with extensions in lime green; purple, mitochondrial-specific ribosomal proteins that are also present in yeast; yellow, mitoribosomal proteins without homologues in yeast.

Extended Data Figure 5 Protein structures in the 39S subunit.

a, mL37 (green) and mL65 (MRPS30; blue) bound to the 39S subunit (16S rRNA backbone in orange; other mitoribosomal proteins in grey). b, mL37 and mL65 (MRPS30) form a pseudo-dimeric assembly on the 39S subunit. c, Superposition of mL37 and mL65 (MRPS30), revealing their common core fold with additional extensions specific to each protein. d, Segment of the mL65 (MRPS30) structure shown in the cryo-EM map (only density corresponding to the selected structural element is shown). Side chain densities can be clearly identified and allowed the identification of the register of the mL65 (MRPS30) sequence in the map. e, Two CX-MS crosslinks between mL65 (MRPS30) and mL37 confirm the placement of mL65 (MRPS30) on the 39S subunit (red, DSS crosslink between lysines; gold, PDH crosslink between carboxyl groups). f, Crosslinks with other mitoribosomal proteins. DSS crosslinks of mL37 with uL2m (purple) and uL29m (gold) and of mL65 (MRPS30) with mL41 (cyan) were observed. All crosslink Cα–Cα distances are 22 Å or shorter. g, Binding site of mL66 (MRPS18A) at the L7/L12 stalk base. mL66 (MRPS18A; gold) and neighbouring mL53 (purple) form a joint β-sheet. h, Side-chain density allows the unambiguous assignment of the location and trace of mL66 (MRPS18A). i, Semi-quantitative MS analysis of protein abundance in the 55S mitoribosome. mL66 (MRPS18A) and mL65 (MRPS30) are present in roughly stoichiometric amounts with other 39S subunit proteins, indicating that only one copy is present per 55S mitoribosome. Only bL12m was detected with clearly more than one copy per 55S mitoribosome, as expected from the multimeric architecture of the L7/L12 stalk58. j, Top view of the CP. In addition to the mitochondrial-specific CP tRNA, several mitochondrial-specific proteins have been recruited to the strongly remodelled CP of the 39S subunit. k, Side view of the CP. Owing to the missing 5S rRNA, the CP is connected to the subunit main body exclusively by protein contacts. l, Structure of the L7/L12 stalk base. Mitochondrial-specific ribosomal proteins mL53, mL54 and mL66 (MRPS18A) have been recruited to the L7/L12 stalk base and may stabilize its connection to the 39S subunit main body.

Extended Data Figure 6 Overall distribution of rRNA and proteins in the mitochondrial 39S subunit and the bacterial 50S subunit.

a, Mitochondrial ribosomal large subunit (subunit interface side on the left, solvent-exposed side on the right). Proteins (cyan) almost entirely cover the solvent-exposed side of the 39S subunit, leaving barely any rRNA (orange) exposed. b, In bacterial ribosomes15 (L1 stalk omitted for clarity), a significant portion of the ribosomal surface on the solvent-exposed side is composed of rRNA (brown), with ribosomal proteins (slate blue) scattered across the subunit surface. The subunit interface side, which makes contact to the small subunit and binds tRNA substrates, is formed by rRNA to a large extent in both types of ribosomes. Landmarks are indicated: L7/L12, L7/L12 stalk base; CP, central protuberance; PTC, peptidyl transferase centre; tunnel exit, polypeptide tunnel exit site.

Extended Data Figure 7 The E-site and the P-site finger of the 39S subunit.

a, 39S subunit (16S rRNA, orange; ribosomal proteins, cyan; uL28m, purple; uL33m, green) with E-site tRNA (blue) modelled based on the superposition of the structure of the bacterial ribosome with bound tRNAs15. The residues corresponding to the tRNA 3′ end binding pocket in bacteria15 are shown in dark red. b, In bacterial ribosomes (23S rRNA, grey; ribosomal proteins, light brown; L1 stalk omitted for clarity, otherwise as in a), rRNA helix 68 (yellow) additionally interacts with the E-site tRNA. This structural element is missing in the 39S subunit. c, The pocket into which the terminal nucleotide of the E-site tRNA inserts in bacterial 23S rRNA (grey)26 is conserved in the mitochondrial 16S rRNA (orange). Ribosomal protein uL28 (light brown) interacts with the E-site tRNA in bacteria15, but is shortened in the 39S subunit (uL28m in purple). d, mL40 (yellow), mL64 (CRIF1, MRPL59) (green) and mL48 (red) approach the area where the E- and P-site tRNAs are located based on superposition of the bacterial structure15. 16S rRNA in orange, 39S proteins in pale cyan. e, Cryo-EM density of the 55S mitoribosome (filtered to 6 Å resolution) segmented into 39S and 28S subunits and A- and P-site tRNAs. The P-site finger is inserted between the A- and P-site tRNAs and contacts both tRNAs. f, Same as e, but shown in top view with part of the ribosome segmented away to reveal the tRNAs.

Extended Data Figure 8 The architecture of the mammalian and yeast mitoribosomal central protuberances.

a, The purine–pyrimidine pattern observed in the additional RNA density at the CP as built model (top) and sequence (bottom). Y, pyrimidines; R, purines; N, undetermined. b, The model of CP tRNA with bases in the segment corresponding to the pattern in a coloured accordingly. c, Simplified secondary structure diagram of mitochondrial tRNAPhe with the segment corresponding to the pattern in a coloured accordingly. d, View of the CP tRNA and the cryo-EM density in the vicinity. Phosphate positions are clearly visible, but the stacked bases merge into continuous strands due to lower local resolution. e, Purines and pyrimidines can be distinguished by their shape. f, Superposition of the large subunit rRNAs of bacterial15 (23S rRNA in grey, 5S rRNA in yellow), mammalian mitochondrial (16S rRNA in orange; CP tRNA in light green) and yeast mitochondrial ribosomes5 (only 84-ES1 shown for clarity, cyan). g, h, Side and top views of the mammalian mitoribosomal CP with the CP tRNA in light green, mL38 in purple, uL18m in blue, mL40 in yellow, mL46 in dark green, mL48 in red and bL31m in orange. i, j, Side and top views of the yeast mitoribosomal CP5 with mL38 in purple, mL40 in yellow, mL46 in dark green, bL31m in orange, uL5m in dark red and 84-ES1 in cyan. Homologous proteins in yeast and mammals occupy roughly similar positions, while mammalian mL48 is positioned similarly to yeast uL5m. Mammalian mL38 is shifted slightly towards the solvent side relative to its yeast counterpart. 84-ES1 occupies a similar position as the anticodon stem-loop part of mammalian CP tRNA, and mL40 binds to the RNA minor groove (star) in both cases. k, l, View of the bacterial ribosomal CP15. 23S rRNA in light grey, 5S rRNA in yellow, uL18 in blue, uL5 in dark red, bL31 in orange, and other ribosomal proteins in light blue.

Extended Data Figure 9 Evolution of RNA binding mitochondrial ribosomal proteins.

a, In the mammalian mitoribosome, the surfaces of mL39 (green) and mL44 (red) corresponding to the RNA binding surfaces of their RNA binding homologues (arrows) are exposed to the solvent. b, The presence of an rRNA expansion segment in the yeast mitoribosome (yeast 21S rRNA5 in orange superimposed on the 39S subunit) explains the arrangement of proteins observed in the mammalian mitoribosome. c, Schematic of the evolution of this region of the mitoribosome. Before the loss of the rRNA expansion segments (ESs), mL39, mL44 and mL57 were possibly simultaneously present in evolutionary precursors of mammalian mitoribosomes.

Extended Data Table 1 CX-MS crosslinks used for confirmation of protein localizations

Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Supplementary Table 1 (see separate file for Supplementary Table 2), and Supplementary References. (PDF 151 kb)

Supplementary Table

This file contains Supplementary Table 2, which shows the results of the mass spectrometric analysis of protein isoforms and variants, the analyzed sequences, and their accession codes. For details, see Legend tab in the spreadsheet file. (XLSX 32 kb)

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Greber, B., Boehringer, D., Leibundgut, M. et al. The complete structure of the large subunit of the mammalian mitochondrial ribosome. Nature 515, 283–286 (2014). https://doi.org/10.1038/nature13895

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