RESEARCH | RESEARCH ARTICLES

RIBOSOME masks during refinement. Rather than use all particles, we combined the data sets that ex- tended to 4.0 Å or better with additional views The structure of the human of the mitoribosome extracted from the remain- ing data sets, giving a total of 449,823 par- ticles (fig. S2A). As the mitoribosome displays mitochondrial preferential orientation on the EM grid, the inclusion of a wider variety of views improved Alexey Amunts,* Alan Brown,* Jaan Toots, Sjors H. W. Scheres, V. Ramakrishnan† the angular sampling of the reconstructions (fig. S2B) and resulted in a higher overall reso- lution than that obtained by using all data. The highly divergent of human mitochondria (mitoribosomes) synthesize Refinement with masks applied over the mt- 13 essential of oxidative phosphorylation complexes. We have determined LSU, mt-SSU, and the head domain improved the structure of the intact mitoribosome to 3.5 angstrom resolution by means of the resolution to 3.3, 3.5, and 3.9 Å, respectively single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected (Fig. 1 and figs. S2 and S3). Although local reso- proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. lution varies within the maps, even at the pe- The head domain of the small subunit, particularly the messenger (mRNA) channel, riphery the density is sufficient for de novo is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, model building (Fig. 1). which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in or . An intrinsic guanosine Overall structure

triphosphatase mediates a contact between the head and central protuberance. The Downloaded from The model of the human mitoribosome contains structure provides a reference for analysis of mutations that cause severe pathologies three rRNA molecules (16S mt-LSU rRNA, 12S and for future . mt-SSU rRNA, and mt-tRNAVal)and80proteins, of which 36 are specific to mitochondria. Addi- itoribosomes have substantially diverged the structure of the intact human mitoribosome tionally, most proteins with homologs in bacteria from bacterial ribosomes, with which they at 3.5 Å resolution by means of electron cryogenic have substantial extensions. The increased pro-

share a common ancestor. They have a microscopy (cryo-EM), revealing an atomic model tein mass results in a ribosome with a distinct http://science.sciencemag.org/ M reversal in their -to-RNA ratio, for the mt-SSU, the unusual dynamics of the morphology (Fig. 2A), a more extensive protein- asaresultofacontractionofribosomal mitoribosome, and the molecular details of protein network of more than 200 contacts (fig. RNA (rRNA) and acquisition of many additional mitochondria-specific bridges between the two S5), and an rRNA core better shielded from re- proteins, which facilitate specific requirements subunits. active oxygen species. of protein synthesis in mitochondria, such as For the mt-SSU, we are able to locate all re- synthesis of hydrophobic proteins and their co- Structure determination ported constituent proteins (30, with 14 specific translational delivery to the membrane (1–3). To overcome the conformational flexibility of to mitochondria), with the exception of MRPS36, The atomic structure of the mitoribosomal large the mt-SSU, we collected 7528 micrographs from confirming the recent observation that it is not subunit (mt-LSU) is known (1–3). However, ob- a total of four data sets, including the data set a mitoribosomal protein (table S2) (5). Addition- taining a similar high-resolution structure of the used to solve the structure of the human mt-LSU ally, mS30 is shown to be present as a single

2 3 entire mitoribosome has been hindered by the ( ). Each data set was initially processed inde- copy in the mt-LSU and renamed to mL65 ( ), on December 19, 2018 conformational variability of the small subunit pendently, with two-dimensional (2D) and 3D and two further homologs of bS18 to that ob- (mt-SSU). The mt-SSU binds messenger (mRNA), classification used to remove particles that aligned served in the mt-LSU are identified (fig. S6). For is involved in accurate initiation and decoding, poorly or corresponded to “free” mt-LSU. The the mt-LSU, we located two additional proteins, and undergoes large-scale conformational changes resultant 884,122 particles were classified further, bL31m and mL54, which were previously iden- during the elongation cycle (4). Here, we report revealing three distinct subpopulations, each re- tified in the porcine mt-LSU (3)andfurtherim- solved to better than 5 Å resolution, in which proved the model at the central protuberance the mt-SSU adopts different orientational states and subunit interface (6). Medical Research Council (MRC) Laboratory of Molecular (fig. S1). The mt-SSU lacks homologs of uS4, uS8, uS13, Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. *These authors contributed equally to this work. †Corresponding To improve the quality of the maps further uS19, and bS20. With the exception of uS4, author. E-mail: [email protected] to permit accurate model building, we applied these proteins have not been substituted by

Fig. 1. Map quality. (A) Maps generated by using masked refinement colored by means of local resolution. The outline of the mask applied to the head is shown as a dashed line. Colored circles pinpoint the location on the mt-SSU of the density examples shown in (B) to (D). (B)Densityfor mS27 at the bottom of the mt-SSU. (C) Well-resolved density for mS38 in the core of the mt-SSU. (D) Density for mS29, including the bound GDP at the head.

SCIENCE sciencemag.org 3APRIL2015• VOL 348 ISSUE 6230 95 RESEARCH | RESEARCH ARTICLES mitochondria-specific elements (Fig. 2B). In can swivel by a further 6° to 7° (13). We observed bridges consist mainly of conserved RNA-RNA bacteria, uS13 and uS19 from the head form only slight rotational movement of the head do- interactions (16). In contrast, the interface of the bridges with the central protuberance and in- main of the mt-SSU rRNA (1° to 2°), which may mitoribosome has a far greater ratio of protein- teract with the anticodon stem loop of tRNAs result from additional protein elements con- mediated contacts, with three protein-protein in the pretranslocation state (7). Alternative necting the head and body, particularly mS37 and six protein-RNA bridges. The effect of the bridgeshavebeenformedbymitochondria- and uS5m. mS29, which forms mitochondria- extensively remodeled bridges is an mt-SSU that specific elements, whereas contacts with tRNA specific bridges with the central protuberance can sample more conformational space than the are lost. The mt-SSU is elongated as a result of of the mt-LSU, is displaced by up to 3.4 Å between bacterial ribosome. mitochondria-specific proteins at both extrem- the three classes in a movement independent of Intersubunit bridges B1, B1b/c, B4, B6, and ities (mS29 at the head and mS27 at the bot- the rRNA head orientation (fig. S9B). B8 that are found in bacterial and cytoplasmic tom). mS26 forms a distinctive 170 Å helix that ribosomes are absent from human mitoribo- wraps around the body. Intersubunit bridges somes (Fig. 4 and fig. S10). Based on class 1, The mt-rRNA is about half the size of the The two ribosomal subunits are connected by a which forms the largest interface between sub- bacterial one (Fig. S7). As observed for the mt- number of intersubunit bridges, although their units, we define seven additional mitochondria- LSU, deletion of mt-SSU rRNA segments oc- composition was unclear at lower resolution specific bridges (designated with the prefix curs at sites that can be bridged by short (two to (14, 15). The movement of the two subunits rel- “m”) (Fig. 4, fig. S11, and table S4). The bridges three nucleotides) sections of rRNA (2)(fig.S8). ative to one another, and rearrangement of the occur along the long axis of the mt-SSU from The remaining rRNA adopts conformations con- bridges, is an intrinsic part of the mechanism of the head to the lower body, although they are sistent with those in bacteria, except for h44. the ribosome. In bacteria and eukaryotes, these primarily centered on the central region of the This helix is a universal element of ribosomes that forms several inter-subunit bridges as well Downloaded from as part of the decoding center. In bacteria, the Fig. 2. Overview of lower part of the helix is rigid and anchored by the human mitoribo- bS20,whereasthemiddlepartformsmajorin- some. (A) Proteins tersubunit bridges (B2a, B3, B5, and B6a), and conserved with the upper part is flexible, being involved in tRNA bacteria (blue), ex-

translocation (8). tensions of homolo- http://science.sciencemag.org/ In the human mitoribosome, the conformation, gous proteins (yellow), coordination, and flexibility of the lower part of and mitochondria- h44 have diverged extensively. In part, this may specific proteins (red). be a response to the loss of constraints of main- rRNA is shown in gray. taining a bridge (B6) with mt-LSU rRNA H62, (B) Location of pro- which is absent. The decreased rigidity (as ob- teins in the human servedthroughhighB-factors)isduetolossof mt-SSU. stabilizing interactions with bS20 and base pair rearrangements. In contrast, the movement of the upper part of h44 is likely restricted by the

presence of mS38. on December 19, 2018

Conformational heterogeneity Ribosomes are highly dynamic, with a ratchet- like rotation of the SSU correlated with mRNA- tRNA translocation. We compared the three distinct subpopulations in our sample with the classical, nonrotated bacterial ribosome (9)and with each other (table S3). The majority of hu- man mitoribosomes (class 1, 60%) adopt an ori- entation that is different from the nonrotated state in bacteria. The small subunits are related by a 7.4° rotation around an axis that runs through h44 and h3 (fig. S9A). Class 2 (22%) Fig. 3. Dynamics of closely resembles the fully ratcheted state ob- intersubunit orienta- served for the bacterial ribosome (Fig. 3A) (10). tions. (A and B)Com- It is related to class 1 by a 9.2° counterclockwise parison of mt-SSU rotation (as viewed from the solvent face of the orientations based on mt-SSU), with bridge B3 acting as a pivot, as common superposition seen in bacteria (11). Class 3 (12%) is related of the mt-LSU. Shifts to class 1 by a rotation of 9.5° around the long between equivalent axis of the mt-SSU (Fig. 3B). This conforma- rRNA phosphorus tional mode is observed in eukaryotic, but not atoms and protein Ca bacterial, ribosomes during the elongation cy- atoms in the different cle and termed “subunit rolling” (12). The re- states are color-coded maining 6% of particles aligned poorly and (0 to 20 Å). (A) Inter- potentially represent a continuum of less well- subunit rotation resem- populated states. bling the ratchetlike In bacterial ribosomes alongside the ratchet- mechanism common to all ribosomes. (B) Rotation of the small subunit around its long axis. (C) like subunit rearrangement, the head domain Schematic of mitoribosomal conformational changes.

96 3APRIL2015• VOL 348 ISSUE 6230 sciencemag.org SCIENCE RESEARCH | RESEARCH ARTICLES body. Bridges mB1a and mB1b at the head of tially longer, and the helix that runs parallel to are the only ribosomes that have acquired an the mt-SSU are analogous to bacterial bridges the interface is specific to mitoribosomes be- intrinsic GTPase activity through the guanosine B1a and B1b because they mediate interactions cause it fills a space generated by the absence 5´-triphosphate (GTP)–binding protein, mS29 (19). with the central protuberance of the LSU through of mt-LSU rRNA H62. In mice, mS29 deficiency is lethal in utero with mitochondria-specific proteins. At the lower body, With the exception of the pivot bridge B3 that abnormal, shrunken mitochondria (20). We located mB6 is formed between the N-terminal exten- maintains nearly identical contacts in all observed mS29 to the mt-SSU head close to the subunit sion of bL19m and mS27. classes, the other bridges form dynamic commu- interface (Fig. 5, D and E), with density for a bound Of the mitochondria-specific bridges, mB4 nication pathways (table S5). The bridges at the nucleoside diphosphate (fig. S12). mS29 is in- buries the largest surface area and is formed by head and bottom of the mt-SSU are particularly volved in coordinating two mitochondria-specific an interaction between mS38 and mt-LSU rRNA dynamic and are therefore poorly resolved. In bridges (mB1a and mB1b) with elements of the H71. At the interface, a helical element of mS38 the ratcheted state, a new bridge (mB7) is formed remodeled central protuberance (fig. S11 and runs parallel to the upper part of h44 before a between uL2m and the phosphate backbone of table S4). Structurally, mS29 is similar to P loop– 90° kink, with the N terminus protruding into h23. The rolling movement preserves the upper containing nucleoside triphosphate hydrolases the mt-SSU rRNA (Fig. 5, A and B). Its location part of h44 and therefore its bridges with the and possesses both Walker A and B motifs (res- and structure resemble those of eL41, a short basic mt-LSU. idues 128 to 135 and 249 to 263, respectively) a-helix that forms a eukaryotic-specific bridge that are necessary for catalysis (21), suggesting (eB14) in the cytoplasmic ribosome (17), suggest- A GTPase mediates intersubunit bridges that mS29 is catalytically active. The structure ing that mS38 and eL41 are the products of con- with the central protuberance also reveals that a hydrogen bond between the vergent evolution. Although the length of eL41 In all domains of life, guanosine triphosphatase carbonyl group of GTP and the main chain amide and the contacts it makes with the two sub- (GTPase) factors act on the ribosome at every of Met100 (fig. S12B) is responsible for the spe- units vary among species (18), mS38 is substan- stage of . However, mitoribosomes cificity of the mitoribosome for guanosine over Downloaded from adenosine (19). Thepresenceofguanosine diphosphate (GDP) bound to mS29 at the subunit interface sug- gests that GTPase activity is linked to subunit association, which is compatible with the ob-

servation that although the mt-SSU readily http://science.sciencemag.org/ binds GTP, the intact mitoribosome does not (19) because the g-phosphate would clash with Thr313.

The mRNA channel During translation, the mRNA occupies an RNA- rich groove that encircles the neck of the SSU (22). The human mitoribosome has substantial differ- ences in both the entry and exit sites of this channel compared with other ribosomes (Fig. 6).

In bacterial ribosomes, incoming mRNA passes on December 19, 2018 through a ring-shaped entrance formed by uS3, uS4, and uS5 that is located between the head and shoulder of the SSU. Basic residues from uS3 and uS4 confer helicase-like activity on the ribo- Fig. 4. Distribution of intersubunit bridges. View showing subunit interfaces. Residues that form some that unwinds secondary structure present bridges are shown as spheres. Bridges invariant in all classes are shown in purple, and dynamic bridges in the mRNA (23). In the human mitoribosome, are in red. mB7 is specific to class 2 and shown in teal. the entry site has been remodeled because of the

Fig. 5. Mitochondria-specific features at the subunit interface. (A)h44, mS38, and mS27 coordinate previously unidentified bridges with the mt-LSU. (B) h44 has been extensively remodeled. The upper half is stabilized by mS38, whereas the lower half has greater flexibility (the representation of the phosphate backbone iscoloredandsizedbyBfactor).(C) h44 in bacterial ribosomes. (D)mS29 mediates mitochondria-specific bridges between the central protuberance and head. (E) In monosomes, mS29 is bound to GDP.

SCIENCE sciencemag.org 3APRIL2015• VOL 348 ISSUE 6230 97 RESEARCH | RESEARCH ARTICLES

6. Materials and methods are available as supplementary materials on Science Online. 7. Y. Chen, S. Feng, V. Kumar, R. Ero, Y. G. Gao, Nat. Struct. Mol. Biol. 20, 1077–1084 (2013). 8. M. S. VanLoock et al., J. Mol. Biol. 304, 507–515 (2000). 9. M. Selmer et al., Science 313, 1935–1942 (2006). 10. D. S. Tourigny, I. S. Fernández, A. C. Kelley, V. Ramakrishnan, Science 340, 1235490 (2013). 11. J. A. Dunkle et al., Science 332, 981–984 (2011). 12. T. V. Budkevich et al., Cell 158, 121–131 (2014). 13. S. Mohan, J. P. Donohue, H. F. Noller, Proc. Natl. Acad. Sci. U.S.A. 111, 13325–13330 (2014). 14. M. R. Sharma et al., Cell 115,97–108 (2003). 15. P. S. Kaushal et al., Proc. Natl. Acad. Sci. U.S.A. 111, 7284–7289 (2014). 16. J. Chen, A. Tsai, S. E. O’Leary, A. Petrov, J. D. Puglisi, Curr. Opin. Struct. Biol. 22, 804–814 (2012). Fig. 6. Remodeling of the mRNA channel. (A) A wide entrance to the mRNA channel is formed by 17. A. Ben-Shem et al., Science 334, 1524–1529 (2011). uS3m and uS5m. (B) Overall path taken by mRNA in the human mitoribosome, with mS39 located near 18. W. Wong et al., eLife 3, 03080 (2014). 19. N. D. Denslow, J. C. Anders, T. W. O’Brien, J. Biol. Chem. 266, the channel entrance. (C) mRNA emerges through an exit formed by bS1m, bS21m, and mS37. 9586–9590 (1991). 20. H. R. Kim et al., FASEB J. 21, 188–196 (2007). 21. J. E. Walker, M. Saraste, M. J. Runswick, N. J. Gay, EMBO J. 1, Downloaded from absence of uS4 and deletion of the C-terminal (29) for a homolog of the N-terminal domain of 945–951 (1982). domain of uS3. The new entry site is dominated bS1 (fig. S14). Unlike bacterial bS1, bS1m is 22. G. Z. Yusupova, M. M. Yusupov, J. H. Cate, H. F. Noller, Cell – by an extension to uS5m that partially compen- tightly associated with the ribosome through 106, 233 241 (2001). 23. S. Takyar, R. P. Hickerson, H. F. Noller, Cell 120,49–58 sates for the lack of uS4 and interacts with uS3m extensive interactions with uS2m and bS21m (2005). (Fig. 6A). As a result, the entrance has shifted, (Fig. 6B). In bacteria, bS1 is composed of six 24. S. M. Davies et al., FEBS Lett. 583, 1853–1858 (2009). and its diameter expanded from 9 to 15 Å (fig. OB folds that work in a concerted manner to 25. P. Yin et al., Nature 504, 168–171 (2013). 26. N. Manavski, V. Guyon, J. Meurer, U. Wienand S13, A and B). The interior of the channel is unfold mRNAs for active translation (30). We http://science.sciencemag.org/ 189 239 264 R. Brettschneider, Plant Cell 24, 3087–3105 (2012). lined with basic (Arg ,Lys ,andArg )and can exclude the possibility that bS1m forms a – 229 27. J. Montoya, D. Ojala, G. Attardi, Nature 290, 465 470 aromatic (Phe ) residues from uS5m, which platform for bS1-like assembly because it lacks (1981). might be involved in direct interactions with the essential solvent-exposed helix that forms 28. J. Rabl, M. Leibundgut, S. F. Ataide, A. Haag, N. Ban, Science mRNA (fig. S13C). the interaction with the following OB domain 331, 730–736 (2011). Although the entrance has widened, the aver- (fig. S14D) (31). However, the structural and po- 29. K. Byrgazov, S. Manoharadas, A. C. Kaberdina, O. Vesper I. Moll, PLOS ONE 7, e32702 (2012). age diameter of the channel is still less than that sitional conservation of bS1m with bS1 and a 30. X. Qu, L. Lancaster, H. F. Noller, C. Bustamante, I. Tinoco Jr., of an RNA duplex. Therefore, mRNAs must enter large electropositive patch that faces the emerg- Proc. Natl. Acad. Sci. U.S.A. 109, 14458–14463 (2012). as a single strand. In the proximity of the channel ing mRNA suggest a role in binding RNA (fig. 31. D. Takeshita, S. Yamashita, K. Tomita, Nucleic Acids Res. 42, entrance is a pentatricopeptide repeat (PPR) pro- S14, E and F). 10809–10822 (2014). tein (mS39) bound to the solvent side of the 32. J. A. Caminero, G. Sotgiu, A. Zumla, G. B. Migliori, Lancet Infect. Dis. 10, 621–629 (2010).

Antibiotic sensitivity and effect head (Fig. 6B). This is the largest protein of the on December 19, 2018 of ribosomal mutations 33. K. M. Keeling, D. Wang, S. E. Conard, D. M. Bedwell, Crit. Rev. mt-SSU and comprises a helix-turn-helix array Biochem. Mol. Biol. 47, 444–463 (2012). stretching over 110 Å. Its knockdown results in Aminoglycosides are potent for com- 34. E. Selimoglu, Curr. Pharm. Des. 13, 119–126 (2007). a substantial overall decrease in mitochondrial bating severe bacterial infections (32) and are 35. Y. Qian, M. X. Guan, Antimicrob. Agents Chemother. 53, 24 4612–4618 (2009). protein synthesis ( ). A conserved property of also used for treatment of genetic disorders – 33 36. A. Soriano, O. Miró, J. Mensa, N. Engl. J. Med. 353, 2305 2306 PPR proteins is the ability to bind single-stranded ( ). Mutations in the mt-SSU rRNA gene, par- (2005). RNA (25), including the 5′ ends of mRNA (26). ticularly A1555G and C1494T, have been re- In our structure, the RNA binding motifs closest ported to predispose carriers to aminoglycoside ACKNOWLEDGMENTS to the channel entrance are solvent-exposed and hypersensitivity linked to increased ototoxicity We thank S. Peak-Chew and M. Skehel for mass spectrometry; remain unoccupied. and nephrotoxicity (34). The structure reveals G. McMullan, S. Chen, and C. Savva for help with data collection; J. Grimmett and T. Darling for help with computing; A. Pulk The exit from the channel between the head that these two mutations would reintroduce and J. Cate for scripts to visualize conformational changes; and platform is important for bacterial trans- base pairs to limit the increased flexibility of R. Nicholls for help with the structure comparison features of lation initiation. In bacteria, the Shine-Dalgarno the decoding center and resemble more close- ProSmart; and P. Emsley for help with ligand analysis in Coot. This sequence base pairs with the otherwise flexible ly the aminoglycoside-binding site in bacterial work was funded by grants from the UK MRC (MC_U105184332 ′ 35 to V.R. and MC_UP_A025_1013 to S.H.W.S.), a Wellcome Trust 3 end of SSU rRNA to specify the position of ribosomes (fig. S15A) ( ). Because the clinical Senior Investigator award (WT096570), the Agouron Institute, the start codon (22). Human mt-mRNAs lack a use of aminoglycosides and oxazolidinones and the Jeantet Foundation (V.R.). J.T. was funded by a MRC Shine-Delgarno sequence and are mostly lead- (fig. S15B) is limited by toxicity owing to inhibi- summer studentship (MC_UP_A025_1013). Cryo-EM density erless (27); therefore, the 3′ end of rRNA is not tion of mitoribosomes (34, 36), the structure maps have been deposited with the Electron Microscopy Data Bank (accession numbers EMD-2876, EMD-2877, EMD-2878, expected to be involved in initiation. Indeed, in will aid the rational design of more selective EMD-2879, EMD-2880, and EMD-2881), and coordinates have the mitoribosome this region is deleted, and the compounds. Similarly, the structure provides a been deposited with the (entry code 3J9M). 3′ endofrRNAisstablyassociatedwithmS37. referencefortheanalysisofmitoribosomalmu- A.A. dedicates his share of the work to his mentor N. Nelson. This represents an example of convergent evo- tations that cause severe pathologies (table S6 lution with the , which also and fig. S16). SUPPLEMENTARY MATERIALS does not require a Shine-Dalgarno for initiation, www.sciencemag.org/content/348/6230/95/suppl/DC1 Materials and Methods in which the function of locking the 3′ end of REFERENCES AND NOTES Supplementary Text 28 – rRNA is performed by eS26 ( ). 1. A. Amunts et al., Science 343, 1485 1489 (2014). Figs. S1 to S16 – At the channel exit, there is a protein with a 2. A. Brown et al., Science 346, 718 722 (2014). Tables S1 to S6 – single oligonucleotide-binding (OB) fold that 3. B. J. Greber et al., Nature 515, 283 286 (2014). References (37–63) 4. W. Zhang, J. A. Dunkle, J. H. Cate, Science 325, 1014–1017 is consistent with a low-resolution EM recon- (2009). 21 October 2014; accepted 5 February 2015 struction (EMD-5693) and biochemical data 5. M. Heublein et al., Mol. Biol. Cell 25, 3342–3349 (2014). 10.1126/science.aaa1193

98 3APRIL2015• VOL 348 ISSUE 6230 sciencemag.org SCIENCE www.sciencemag.org/content/348/6230/95/suppl/DC1

Supplementary Material for

The structure of the human

Alexey Amunts, Alan Brown, Jaan Toots, Sjors H. W. Scheres, V. Ramakrishnan*

*Corresponding author. E-mail: [email protected]

Published 3 April 2015, Science 348, 95 (2015) DOI: 10.1126/science.aaa1193

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S16 Tables S1 to S6 Full Reference List Supplementary,information, ! Electron)microscopy) Human! mitoribosomes! were! prepared! as! previously! described! (2).! Aliquots! of! 3! μl! of! purified!mitoribosomes!at!a!concentration!of!~80!nM!(0.23!mg/ml)!were!incubated!for! 30!s!on!glowDdischarged!holey!carbon!grids!(Quantifoil!R2/2),!onto!which!a!homeDmade! continuous!carbon!film!(~50!Å!thick)!had!been!deposited.!Grids!were!blotted!for!2.5!s!in! 100%!ambient!humidity!and!flash!frozen!in!liquid!ethane!using!an!FEI!Vitrobot.!Grids! were!transferred!to!an!FEI!Titan!Krios!electron!microscope!operated!at!300!kV.!Images! were! collected! using! FEI’s! automated! single! particle! acquisition! software! (EPU)! and! recorded! on! a! backDthinned! FEI! Falcon! II! detector! at! a! calibrated! magnification! of! 104,478!(yielding!a!pixel!size!of!1.34!Å).!An!inDhouse!system!(37)!was!used!to!intercept! the!videos!from!the!detector!at!a!rate!of!17!frames!for!each!1!s!exposure.!Defocus!values! ranged!from!1.5D3.5!µm.!All!datasets!were!collected!under!identical!conditions.!

Initial)image)processing) Contrast! transfer! function! parameters! were! estimated! using! CTFFIND3! (38).! Particles! were! picked! using! semiDautomated! particle! picking! with! RELION! (39).! All! 2D! and! 3D! classifications!and!refinements!were!performed!using!RELION!(39).!We!used!referenceD free!2D!class!averaging!to!discard!bad!particles,!leaving!a!total!of!884,122!particles.!3D! classification!of!all!particles!was!used!to!isolate!mitoribosome!subpopulations,!with!each! class!subsequently!refined.!3D!refinements!were!initiated!with!a!60!Å!lowDpass!filtered! cryoDEM!reconstruction!of!the!yeast!mitoribosome.!BeamDinduced!particle!movements! were!corrected!using!statistical!movie!processing!in!RELION!(37,)40).!

Masked)refinement)) To!improve!the!quality!of!the!maps!for!both!the!mtDSSU!and!mtDLSU!we!firstly!initiated! independent! refinement! of! each! of! the! four! collected! datasets.! The! first! dataset! represents!a!reprocessing!of!the!data!used!in!(2).!The!resolutions!for!each!dataset!were! 3.7,!4.0,!4.4!and!4.6!Å.!The!best!two!datasets!(3.7!and!4.0!Å)!were!combined!(286,106! particles)! and! the! resultant! 3D! reconstruction! analyzed! for! angular! distribution.! As! some!orientations!had!more!particles!assigned!than!others!(as!a!result!of!preferential! orientation!of!the!mitoribosome!on!the!EM!grid),! we! selected! alternative! orientations! from!the!2D!classifications!for!the!other!two!datasets!to!improve!the!angular!sampling.! Combinations! of! these! particles! with! the! best! two! datasets! gave! a! total! of! 449,823! particles.!These!data!were!3D!refined!with!soft!masks!applied!(1)!over!the!mtDLSU!and! mtDSSU!and!the!head!domain!of!the!mtDSSU.!For!the!mtDSSU,!the!resolution!of!the!final! reconstruction!containing!these!additional!particles!(3.49!Å)!was!at!a!higher!resolution! than!excluding!the!data!(3.57!Å)!or!including!all!particles!from!all!datasets!(3.54!Å).!The! nominal!resolution!for!the!mtDLSU!was!3.3!Å!and!for!the!masked!head!of!the!mtDSSU,! 3.9!Å.!! ! Prior! to! visualization,! all! density! maps! were! corrected! for! the! modulation! transfer! function! (MTF),! and! then! sharpened! by! applying! a! BDfactor! (Table! S1)! that! was! estimated!using!automated!procedures!(41).!!! ! Model)building) The!revised!Cambridge!Reference!Sequence!(rCRS)!(42)!is!used!to!number!the!mtDNAD encoded!nucleic!acid!components!of!the!mitoribosome.!Ribosomal!proteins!are!named! following!the!new!nomenclature!(43)!and!rRNA!helices!are!numbered!corresponding!to! those!in!Escherichia)coli!(44).! ! The!better!resolved!density!for!the!mtDLSU!allowed!a!number!of!improvements!to!our! previously! published! model! (2).! For! some! regions,! the! structure! of! the! porcine! mitoribosome! was! used! to! guide! model! building! (3).! Improvements! included! the! identification!of!mL54!(Uniprot!ID:!Q6P161)!at!the!L7/L12!stalk!and!the!extension!of!the! termini!of!mL62!(ICT1)!and!the!NDterminus!of!mL42.!A!number!of!additional!features! were!also!present!at!the!subunit!interface;!the!NDterminus!of!bL19m!that!faces!the!mtD SSU,!a!loop!of!bL19m!(residues!198D212),!the!tip!of!rRNA!H67!which!forms!bridge!B7a,! and! a! region! of! uL2m! (residues! 170D175)! that! contributes! to! mitochondriaDspecific! bridge!mB2.!The!density!for!the!central!protuberance!was!also!much!better!resolved;! this!allowed!identification!of!bL31m!(Uniprot!ID:!Q7Z7F7)!and!rebuilding!and!extension! of!mL40,!mL46!and!mL48.!Additional!magnesium!ions!were!also!identified!(Table!S1).! Additionally,!the!density!for!the!nascent!polypeptide!in!the!exit!tunnel!is!improved.!! ! Refinement) For!optimal!fitting!of!the!model!into!the!EM!density!map,!we!used!REFMAC!v5.8!adapted! for!EM!refinement!(1)!to!refine!each!subunit!model!against!its!masked!map.!To!optimize! stereochemistry!at!the!subunit!interface,!the!intact!mitoribosome!was!refined!against!a! composite!map!generated!by!combining!the!three!masked!maps!(mtDLSU,!mtDSSU!and! for!the!mtDSSU!head).!Intrasubunit!external!restraints!were!generated!with!ProSMART! (45)! using! the! models! of! the! mtDSSU! and! mtDLSU! that! had! been! refined! against! the! highestDresolution! data.! Restraints! were! not! generated! for! the! subunit! interface! to! encourage!conformational!independence.!To!analyze!the!different!conformation!states! of!the!human!mitoribosome,!the!subunits!were!independently!fit!into!the!maps!for!each! state! using!Chimera!(46),! and! largeDscale!conformational!changes!within!each!subunit! were!manually!adjusted!using!rigidDbody!fitting!in!Coot!(47).!! !

FSCaverage!was! monitored! using! during! refinement! to! follow! the! fitDtoDdensity,! and! the! final! model! was! validated! using! MolProbity! (48).! CrossDvalidation! against! overDfitting! was! calculated! as! previously! described! (1,) 49)! and! refinement! statistics! are! given! in! table!S1.! ! Figures) All! figures! were! generated! using! PyMOL! (50),! Chimera! (46)! or! QuteMol! (51).! Local! resolution! representations! were! created! with! ResMap! (52).! The! secondary! structure! diagram! of! the! mtDSSU! rRNA! was! modified! from! the! Comparative! RNA! website! (53)! using!base!pairs!information!extracted!from!the!model!using!DSSR!(54).!! ) ) Table& S1.& Refinement( and( model( statistics( for( the( individual( subunits( and( intact( 55S( mitoribosome.( The( mitoribosome( was( refined( against( a( composite( map( formed( from( masked(maps(for(mt

Data&Collection& & Particles( 449,823( & Pixel(size((Å)( 1.34( & Defocus(range((µm)( 1.5(–(3.5( & Voltage((kV)( 300( & Electron(dose((e<(Å<2)( 25( & mt2SSU& mt2LSU& 55S& Model&composition( & Non

& FSCaverage( 0.88( 0.86( 0.85( Rms&deviations( & Bonds((Å)( 0.0076( 0.0075( 0.0064( & Angles((°)( 1.42( 1.47( 1.22( Validation&(proteins)( 2.8((93rd( 2.6((96th( 2.5((98th( & Molprobity(score( percentile)( percentile)( percentile)( 5.8((100th( 5.2((100th( 4.2((100th( & Clashscore,(all(atoms( percentile)( percentile)( percentile)( & Good(rotamers((%)( 86.6( 89.8( 91.2( Ramachandran&plot( & Favored((%)( 87.4( 88.1( 89.4( & Outliers((%)( 3.3( 3.1( 2.4( Validation&(RNA)( & Correct(sugar(puckers((%)( 97.1( 95.9( 97.2( Good(backbone( & 68.0( 65.3( 66.5( conformations((%)( ( Table&S2.!Mitochondrial!ribosomal!proteins!(MRPs)!of!the!human!mt8SSU.!!Protein!residues!and!signal!!positions!are!numbered!according!to! UniProt! (55)! with! updated! signal! peptide! cleavage! positions! based! on! the! structure.! Mitochondria8specific! proteins! were! compared! to! known! structures!in!the!PDB!using!PDBeFold!(56)!and!DALI!(57).! ! Mature& protein& MW&(excl.& Built&residues& UniProt& Chain& (range,&amino& signal&peptide,& (range,&amino& MRP& Alias& ID& ID& acids)& kDa)& acids)& Notes& bS1m! MRPS28! Q9Y2Q9! W! 72!–!187! 13.1! 778173! OB!motif.!Structural!homolog!of!the!N8 terminal!domain!of!bS1.! uS2m! MRPS2! Q9Y399! B! 1!–!296! 33.2! 588274! ! uS3m! MRPS24!! Q96EL2! C! 36!–!167! 15.3! 368167! ! uS5m! MRPS5! P82675! D! 1!–!430! 48.0! 888107;!1268150;! ! 1548430! bS6m! MRPS6! P82932! E! 2!–!125!! 14.1! 28123! ! uS7m! MRPS7! Q9Y2R9! F! 38!–!242!! 24.1! 35853;!618242! ! uS9m! MRPS9! P82933! G! 1!–!396!! 45.8! 718176;!1988396! ! uS10m! MRPS10! P82664! H! 1!–!201!! 23.0! 618182! ! uS11m! MRPS11!! P82912! I! 1!–!194! 20.6! 598194! ! uS12m! MRPS12! O15235! J! 30!–!138! 12.1! 318138! ! uS14m! MRPS14! O60783! K! 1!–!128!! 15.1! 288128! ! uS15m! MRPS15! P82914! L! 58!–!257! 23.5! 668229! ! bS16m! MRPS16! Q9Y3D3! M! 35!–!137! 11.5! 108125! Zn8binding! uS17m! MRPS17!!! Q9Y2R5! N! 21!–!130!! 12.3! 58111! ! bS18b! MRPS1882!!! Q9Y676! O! 1!–!258! 29.4! 528236! Zn8binding! (mS40)! bS18c! MRPS1881!! Q9Y3D5! P! 1!–!142! 15.8! 478142! Zn8binding! (bS18m)! bS21m! MRPS21!!! P82921! Q! 1!–!87! 10.7! 2887! ! mS22! MRPS22! P82650! R! 1!–!360! 41.2! 678308! ! mS23! MRPS23! Q9Y3D9! S! 1!–!190! 21.8! 28127! ! mS25! MRPS25! P82663! T! 1!–!173! 20.1! 28163! Thioredoxin8like!fold.!! mS26! MRPS26! Q9BYN8! U! 28!–!205! 21.3! 278199! ! mS27! MRPS27!! Q92552! V! 1!–!414! 47.6! 368269;!2788291;! Pentatricopeptide!repeat!proteins!! 3128345;!3558400! mS29! MRPS29! P51398! X! 19!–!398! 43.4! 518168;!1778195;! Structurally!similar!to!P8loop!NTPases.! 2208398! GDP!bound.! mS31! MRPS31! Q92665! Y! 66!–!395! 38.1! 2768383! ! mS33! MRPS33! Q9Y291! Z! 2!–!106! 12.5! 5891! ! mS34! MRPS34! P82930! 0! 2!–!218! 25.5! 68154;!1628213! ! mS35! MRPS35!! P82673! 1! 1!–!323! 36.8! 528270;!2878323! Three!domains;!the!NT!domain!contacts! mS39.!The!middle!domain!has!structural! similarity!to!peptidyl8tRNA!hydrolases! including!ICT1,!and!YaeJ!and!forms!a! shared!β8sheet!with!uS10.!The!C8terminal! domain!binds!mS29.!! mS37! MRPS37! Q96BP2! 2! 1!–!118! 13.5! 28117! ! (CHCHD1)! mS38! MRPS38! Q9NWT8! 3! 1!–!199! 22.4! 1288196! ! (AURKAIP1)!! mS39! MRPS39! Q96EY7! 4! 38!–!689! 74.4! 568133,!helices! Pentatricopeptide!repeat!protein.! (PTCD3)!! ! ! ! Table&S3.!Comparison!of!mt-SSU!rotations!based!on!common!superposition!of!the!mt- LSU.! PDB! ID:! 2J00! was! used! as! the! unrotated! form! (9)! and! PDB! ID:! 4JUW! as! the! ratcheted!form!(10)!of!the!bacterial!ribosome.!The!screw!axes!and!degree!of!rotation!(°)! were!calculated!with!ProSMART!(58).!! ! ! ! Bacterial&ribosome& Human&mitoribosome&

! Unrotated& Rotated& Class&1& Class&2& Class&3&

Unrotated& ! 8.0! 7.4! 9.0! 14.4!

Rotated& 8.0! ! 9.7! 0.8! 10.5!

Class&1&& 7.4! 9.7! ! 9.2! 9.5!

Class&2& 9.0! 0.8! 9.2! ! 8.8!

Class&3& 14.4! 10.5! 9.5!! 8.8! ! Table&S4.!Residues!involved!in!intersubunit!bridges.!The!analysis!is!based!on!class!1!with!the!exception!of!bridge!mB7,!which!is!specific!to!class!2.!An! analysis!of!bridge!reAarrangement!between!the!three!classes!is!given!in!Table!S5.!! ! Bridge& Interaction& mt7SSU& Residues& mt7LSU& Residues& Notes& type& component& component& B2a! RNA:RNA! rRNA!h44! 1490A1493;!1559A rRNA!H69! 2576;!2581A2583! ! 1562;!1582! B2b! RNA:RNA! rRNA!h24! 1071A1073! rRNA!H67! 2547A2548;!2590A ! 2592! B2c! RNA:RNA! rRNA!h20! 1058A1061! rRNA!H66;!! 2511A2514;! ! rRNA!H67! 2541A2543! B3! RNA:RNA! rRNA!h44! 1502A1504;!1548A rRNA!H71! 2610A2613;! ! 1550! 2621A2624! B5! Protein:RNA! rRNA!h14! 811A812! uL14m! 43;!118;!120! ! B7a! RNA:RNA! rRNA!h23! 1000A1001! rRNA!H68! 2558A2559! ! B7b! Protein:RNA! rRNA!h24! 1062A1063! uL2m! 231! ! mB1a! Protein:protein! mS29! 231A234! mL46! 98A120!(partially! Unbuilt!residues!168A177!from! modeled!loop)! mS29!may!also!contribute.! mB1b! Protein:protein! mS29! 276A279;! mL48! 138A147! ! Protein:RNA! rRNA!h42! 1395A1396;! mL40! (unmodeled)! rRNA!h42! 1412A1414! CAterminal!helix! (194A201)! mB2! Protein:protein! bS6m! 24;!84A86! uL2m! 171A174! ! mB3! Protein:RNA! rRNA!h27! 1153A1154!! rRNA!H67! 2543A2544! ! mB4! Protein:RNA! mS38! 151A152;!! ! 2595A2597;! ! 155A156;! rRNA!H71! 2622A2623! 159;!163! rRNA!H67! 2638A2640;! mB5! RNA:RNA! rRNA!h44! 1505;!1539! uL14m! 50A52! ! mB6! Protein:protein! mS27! 51;!87;!90;!93;! bL19m! NAterminus! Poorly!resolved;!highly!flexible! 131;!134A135! mB7! Protein:RNA! rRNA!h23! 981A982! uL2m! 218! Specific!to!class!2.! & Table&S5.!Bridge!rearrangements!for!each!subpopulation!relative!to!class!1.!! ! Bridge& Class&2& Class&3& B2a! Preserved.! Preserved.! B2b! Contact!broken.!rRNA!h24!(bases!1072A1074)!has!moved!by!6Å.! Contact!broken.!rRNA!h24!(1072A1074)!has!moved!by!5Å.! Contact!broken.!Bases!1059A1061!have!shifted!by!6A7Å.!Possible! B2c! Contact!broken.!Bases!1059A1061!have!shifted!by!6A7Å.! new!MgAcoordinated!bridge!between!1057A1058!and!2543A2544.! B3! Unchanged.! Unchanged.! A!~7Å!shift!of!h14!results!in!closer!contacts!between!the!rRNA!and! ~3Å!shift!of!h14!results!in!closer!contacts!between!the!rRNA!and! B5! uL14m.! uL14m.! B7a! Contact!broken.!9Å!shift!of!h23.! Contact!broken.!8Å!shift!of!h23.! B7b! Contact!broken.!Almost!8Å!shift!of!h24.! Contact!broken.!8Å!shift!of!h24.! mB1a! Bridge!has!opened!and!nature!of!contacts!changed.! Preserved!despite!movement!of!mS29.! mB1b! mS29!no!longer!contributes!to!the!bridge!due!to!a!17Å!movement.! Preserved.! mB2! Widened!by!3Å.! Broken.!12Å!movement!of!bS6m.! mB3! Preserved.! Bridge!opened!by!almost!4!Å.! mB4! Preserved.! Preserved.! mB5! Preserved.! Preserved.! mB6! Movement!of!mS27!towards!bL19m.! Density!is!unclear.! mB7! Bridge!specific!to!class!2.! Absent.! ! & & & & & & & & & Table&S6.!Mutations!in!MRPs!of!the!mtASSU.!!! !

MRP! Mutation! Clinical&significance! References! Possible&Effect! bS16m! R111X! Corpus!callosum! (59)! Nonsense!mutation!results!in!premature!degradation!of!bS16m.! agenesia,!hypothonia! and!fatal!neonatal! lactic!acidosis! mS22! R170H! Edema,! (60)! Mutation!R170H!in!mS22!might!sterically!disturb!the!interprotein!ßA cardiomyopathy,! sheet!arrangement!that!forms!binding!site!of!mS22!onto!bS16m.! tubulopathy,!and! hypotonia! mS22! L215P! Cornelia!de!LangeA (61)! Mutation!results!in!premature!degradation!of!mS22.!mS22!helps! like!syndrome! stabilize!uS5m!at!the!mRNA!channel!entrance.! oedema,! cardiomyopathy!and! tubulopathy! ! ! !! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Fig.!S1.!In#silico!processing!of!mitoribosome!datasets.!A.!After!initial!removal!of!poorly! aligned!particles!and!‘free’!mt8LSU!by!2D!and!3D!classification,!a!subsequent!round!of! 3D! classification! with! the! retained! particles! from! all! datasets! reveals! three! distinct! conformations!of!the!human!mitoribosome!(classes!183,!sorted!by!population!size)!that! are!colored!by!local!resolution.!Class!4!represents!particles!that!aligned!poorly.!B.!‘Gold8 standard’!FSC!plots!for!the!three!classes.!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Fig.! S2.! To! improve! the! resolution! of! the! maps! for! model! building! we! utilized! map! refinement! using! masked! maps.! Each! dataset! was! initially! processed! separately! as! described!in!(2).!This!resulted!in!four!maps!that!reached!nominal!resolutions!of!3.7,!4.0,! 4.4!and!4.6!Å.!The!two!datasets!at!highest!resolution!were!combined!and!supplemented! with! views! extracted! from! 2D8classification! of! the! remaining! datasets! (c! and! d)! that! were! poorly! represented! in! the! first! two! datasets.! 3D! refinement! of! these! data! with! masks! applied! over! the! mt8LSU,! mt8SSU! and! head! region! improved! the! resolution! of! these!maps!to!3.3,!3.5!and!3.9!Å,!respectively.!Local!resolutions!for!these!masked!maps! are!given!in!Fig.!1.!B.!Projection!of!the!first!two!euler!angles!on!a!sphere!in!an!arbitrary! orientation.! Each! point! of! the! sphere! represents! a! different! projection! direction.! The! length! of! the! cylinder! represents! how! many! individual! particles! are! assigned! to! that! direction.! There! is! a! linear! scale! for! both! the! height! of! the! cylinder! from! low! to! high! number! of! particles! and! from! blue! to! red.! The! angular! distribution! improves! when! datasets!a!and!b!(top)!are!supplemented!with!additional!2D!classes!from!datasets!c!and! d!(bottom).!C.!FSC!curves!for!the!masked!maps.!! ! ! ! ! Fig.!S3.!The!local!resolution!of!the!mt8SSU!head!is!improved!by!applying!masks!during! refinement! (see! Fig.! 1).! A.! View! of! the! head! region! of! the! mt8SSU! of! the! intact! mitoribosome!(class!1)!without!masking!reveals!that!the!head!is!poorly!resolved!due!to! conformational! variability.! B.! Masking! of! the! mt8SSU! improves! the! local! resolution! to! approximately!485!Å.!C.!Subsequent!masking!of!only!the!head!domain!improves!the!local! resolution!further!to!aid!model!building!of!this!region,!particularly!for!mS39.!! ! ! ! Fig.! S4.! Overfitting! was! monitored! using! cross8validation! (1,# 49).! Fourier! shell! correlation!curves!were!calculated!between!the!refined!model!and!the!final!map!(black),! along! with! the! self! (FSCwork,! blue)! and! cross8validated! (FSCtest,! red)! correlations.! FSC! curves! for! mt8LSU! (A)! and! mt8SSU! (B)! demonstrate! that! no! overfitting! of! the! final! models!is!apparent.!! ! ! ! ! ! !

! ! ! Fig.! S5.! Interactions! between! mitoribosomal! proteins! of! the! mt8SSU.! A.! Surface! representation!showing!interaction!between!mitoribosomal!proteins!with!homologs!in! bacteria!(blue)!and!mitochondria8specific!proteins!(red).!B.!Protein8protein!network!of! A.! The! node! size! represents! the! relative! molecular! mass! of! the! protein! and! the! edge! thickness!the!solvent!accessible!surface!buried!in!the!interface.!Interactions!conserved! with! bacteria! are! shown! in! black,! and! mitochondria8specific! interactions! in! red.! The! protein8protein!network!for!the!mt8LSU!is!given!in!((ref.#2).! ! ! ! ! ! Fig.! S6.!The!human!mitoribosome!contains!three!structural!homologs!of!bS18;!one!in! the! mt8LSU! and! two! in! the! mt8SSU.! While! the! Zinc! (green! sphere)! binding! motif! is! conserved!the!N8!and!C8termini!extensions!do!not!show!any!conservation.!!! ! ! ! ! ! Fig.! S7.! A.! Surface! representation! showing! the! contraction! of! mt8SSU! rRNA.! rRNA! present! in! both! bacteria! and! human! mitoribosomes! is! shown! in! black,! and! excised! sections!in!white.!Proteins!absent!from!the!mitoribosome,!but!observed!in!bacteria!are! shown!in!blue.!B.!Secondary!structure!representation!of!A.!! ! ! Fig.!S8.!Secondary!structure!diagram!for!mt8SSU!rRNA!colored!by!domain.!Sections!that! bridge! conserved! regions! are! highlighted! in! red.! Unbuilt! regions! are! shown! in! blue! lettering.! ! ! ! ! ! Fig.! S9.!A.!Based!on!common!superposition!of!the!large!subunits,!mt8SSU!is!related!to! the!bacterial!SSU!in!the!unrotated!form!(9)!by!a!7.4°!rotation!around!an!axis!that!runs! through!h44!and!h3!(black!line).!B.!Superposition!of!the!mt8SSU!from!classes!1!and!2! reveals!that!the!rRNA!head!domains!align!with!very!little!movement.!Independent!of! rRNA!head!movement,!the!mS29!and!the!C8terminal!domain!of!mS35!move!by!3.4!Å!that! alters!the!contacts!with!central!protuberance!of!the!mt8LSU.!Class!1!is!shown!in!grey,! and!class!2!in!color.!! ! ! Fig.!S10.!Bridges!conserved!with!bacterial!and!cytoplasmic!ribosomes.!! ! ! ! ! ! Fig.! S11.!Intersubunit!bridges!specific!to!mitoribosomes.!mB7!is!only!observed!in!the! ratcheted!form!(class!2).!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Fig.! S12.! A.! mS298bound! GDP! showing! map! density! (mesh).! B.! Environment! of! the! bound!GDP.!A!hydrogen!bond!between!the!carbonyl!group!of!the!guanosine!moiety!and! the!main!chain!amide!of!Met100!contributes!to!the!specificity!for!GDP!over!ADP.!! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Fig.!S13.!A.!The!channel!entrance!in!bacteria.!!uS3!and!uS4!confer!helicase8like!activity! on!the!ribosome.!B.!The!channel!entrance!in!the!human!mitoribosome!is!wider!due!to! the! absence! of! uS4! and! the! N8terminal! domain! of! uS3.! ! A! mitochondria8specific! extension! of! uS5m! partially! fills! the! void! left! by! uS4.! C.! uS5m! lays! out! the! channel! interior! with! basic! (Arg189,! Lys239,! Arg264)! and! aromatic! (Phe229)! residues,! which! might!be!involved!in!direct!interactions!with!mRNA.!!! ! !

! ! Fig.!S14.!A.!The!location!of!bS1m!is!compatible!with!a!low8resolution!map!of!bS1!bound! to!the!bacterial!ribosome!(EMD85693).!B.!A!view!from!above!as!indicated!in!A.!C.!bS1m! is! tightly! bound! to! the! mitoribosome! through! extensive! interactions! with! uS2m! and! mS23.! D.! Superposition! of! bS1m! with! the! N8terminal! domain! of! bS1! (PDB! ID:! 4Q7J).! bS1m! lacks! the! C8terminal! helix! that! connects! consecutive! OB8folds! in! bS1.! E.! Surface! representation!of!bS1m,!bS21m!and!mS37!showing!electrostatic!potential.!The!surface! of!bS1m!facing!the!channel!and!the!channel!exit!(F)!are!electropositive.! ! ! ! ! ! Fig.! S15.! A.! Paromomycin! is! a! member! of! the! aminoglycoside! class! of! antibiotics.! It! binds! adjacent! to! the! A! site! of! the! decoding! center! of! the! bacterial! rRNA! (grey)! and! induces!codon8anticodon!misreading!and!prevents!translocation!of!mRNA:tRNA!to!the!P! site.!The!binding!site!in!the!human!mt8rRNA!(blue)!is!more!flexible!due!to!loss!a!G:C!base! pair.! Mutations! in! three! residues! surrounding! the! binding! site! would! induce! a! more! bacteria8like!conformation!and!are!implicated!in!aminoglycoside!hypersensitivity!(Table! S6).!B.!The!binding!pocket!near!the!A!site!in!the!mt8LSU!rRNA!(blue)!for!oxazolidinones! (including!linezolid)!is!nearly!identical!to!that!observed!in!bacteria!(shown!in!grey)!(62,# 63).!In!both!panels,!antibiotics!are!shown!in!yellow.!! ! ! ! ! Fig.! S16.! Mutations! in! the! mt8SSU! proteins! bS16m! and! mS22! are! associated! with! pathological!phenotypes!(Table!S3).!A.!bS16m!and!mS22!are!located!on!the!solvent!side! of!the!mt8SSU!body!and!are!tightly!associated!through!a!shared!ß8sheet.!Both!a!nonsense! mutation!in!bS16m!(Arg111Stop)!and!a!missense!mutation!in!mS22!(Leu215Pro)!result! in! premature! degradation! of! the! protein! product! (59,! 61).! Absence! of! either! protein! would!result!in!a!destabilization!of!the!lower!body,!including!uS5m!that!forms!part!of! the!mRNA!channel!entrance.!B.!Mutation!Arg170His!in!mS22!might!sterically!hinder!the! formation!of!the!interprotein!ß8sheet!with!bS16m.!

!

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