Source: https://amuntslab.org/wp-content/uploads/2020/09/2017-5.pdf Primary article: PMID:28154081. Downloaded 2026-09-08; extracted by pdftotext -layout. Species: Saccharomyces cerevisiae. R ES E A RC H ◥ of the 24 mt-tRNAs encoded by the yeast mito- REPO R T chondrial genome. The ribosomal proteins are named in accordance with the nomenclature adopted by the ribosomal community (9), with the RIBOSOME standard yeast name according to the Saccharo- myces Genome Database (10) in parentheses. The structure of the yeast The 15S rRNA of the mt-SSU is longer than the equivalent 16S rRNA in Escherichia coli (1649 compared to 1542 nucleotides). Of these, 1501 mitochondrial ribosome nucleotides have been modeled, revealing an rRNA core that adopts a domain structure sim- ilar to that of 16S rRNA (fig. S5). Discrepancies Nirupa Desai,1 Alan Brown,1 Alexey Amunts,1,2 V. Ramakrishnan1* occur mostly at the periphery of the mitoribo- some: Helices h6, h8, h17, h21, h33, and h39 are Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of shorter than their bacterial equivalents, but the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we this is counteracted by three rRNA expansion have determined the structure of the 75-component yeast mitoribosome to an overall segments (h16-ES, h17-ES, and h41-ES) and ex- resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and tensions of h9, h44, and of the 5′ and 3′ tails. includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the Many of the 148 nucleotides that could not be evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, modeled occupy these peripheral expansion seg- including the acquisition of a putatively active enzyme, give the mitoribosome a distinct Downloaded from http://science.sciencemag.org/ on December 19, 2018 ments, which have poor density. Overall, there architecture compared to the mammalian mitoribosome. At an expanded messenger RNA is less expansion of rRNA than in the yeast mt- channel exit, there is a binding platform for translational activators that regulate translation LSU (4) but considerably more than in the 12S in yeast but not mammalian mitochondria. The structure provides insights into the rRNA of human mitoribosomes, which exhibits evolution and species-specific specialization of mitochondrial translation. substantial rRNA reduction (5). M The yeast mt-SSU has an almost complete itochondria are eukaryotic organelles that appears to be on an evolutionary path that has complement of proteins with homologs in the carry out aerobic respiration. Resulting not experienced rRNA contraction (4). Further- bacterial SSU, with only bS20 absent (Fig. 1B). from their likely ancestry as endosym- more, it differs functionally from the mammalian The mammalian mt-SSU lacks four additional bionts (1), mitochondria retain a vestig- mitoribosome by synthesizing a soluble protein homologs, suggesting that this loss occurred ial genome of ~3 to 100 genes, depending in addition to integral membrane proteins. The after the mitoribosomes diverged from a com- on the species. All mitochondrial DNA (mtDNA) structure of the yeast mt-LSU showed a rerouted mon ancestor. The yeast mitochondrial homo- encodes at least some of the essential trans- polypeptide exit channel that may be a response log of uS3 (uS3m; Var1) is the only soluble membrane subunits of the oxidative phospho- to this requirement (4). protein encoded by mtDNA (11, 12). However, rylation complexes. To synthesize these proteins, However, without high-resolution structural in- unlike nuclear-encoded uS3m in other species, mitochondria have dedicated ribosomes (mito- formation for complete mitoribosomes of non- yeast uS3m (Var1) has a ~30% asparagine con- ribosomes). Nearly all mitoribosomal proteins mammalian species, it has not been possible to tent. These asparagine residues are distributed and all translational factors are encoded by observe the effects of distinct evolutionary pressures in solvent-exposed regions throughout the pro- nuclear DNA and imported from the cytoplasm, and species-specific mitochondrial translation on tein (Fig. 2B). The high asparagine content is whereas mtDNA encodes the mitoribosomal RNA the mitoribosomal small subunit (mt-SSU). Re- likely a response to the propensity of mitochon- (rRNA) and mitochondria-specific transfer RNAs cognized selective pressures on the mt-SSU include drial genomes to be AT rich (yeast mtDNA has (mt-tRNAs). mRNA binding, the initiation of translation, and an 83% AT content). Asparagine is the only hy- Mitochondrial translation displays consider- decoding. Notably, translational initiation in yeast drophilic residue specified by a codon formed able species-specific specialization, largely dic- mitochondria involves long 5′ untranslated regions exclusively of adenine and thymine nucleotides tated by the translational requirements of the (5′ UTRs) of mitochondria-encoded mRNAs and (AAT). Consistent with the AT content determin- mitochondrial genome (2). This specialization transcript-specific translational activators, neither ing the preferred amino acid, only 2 of the 127 manifests in the diverse compositions and struc- of which occurs in mammalian mitochondria (8). asparagine residues in uS3m (Var1) are encoded tures of mitoribosomes, which are distinct from To improve our understanding of the effect of by the alternative AAC codon. one another and from all known ribosomes des- species-specific mitochondrial translation on mito- The yeast mt-SSU also contains 14 mitochondria- pite sharing an ancestor with modern bacterial ribosomal diversity, we have solved the structure specific proteins, of which 7 have homologs in ribosomes (2, 3). of the complete yeast mitoribosome by electron the mammalian mitoribosome and 7 are specific Our current understanding of this diversity cryomicroscopy (Fig. 1A and fig. S1). The yeast to the yeast mitoribosome. mS38 (Cox24) had at the atomic level is limited to comparisons mitoribosome adopts three well-populated con- not previously been assigned as a yeast mito- of the structure of the large subunit of the formations (classes A to C) that are resolved to ribosomal protein and was identified with a Saccharomyces cerevisiae mitoribosome (mt-LSU) between 3.3 and 5.0 Å resolution (fig. S2A). To sequence obtained directly by interpreting the (4) with the structures of the complete human improve the quality of the maps further, masks density (fig. S6). A role for Cox24 in mitochon- (5) and porcine (6) mitoribosomes. These com- were applied during processing, which allowed drial translation is consistent with a previous parisons have revealed that the major evolu- the mt-LSU to be refined to 3.2 Å, the body of report that Cox24 null mutants have reduced tionary trajectory for mammalian mitoribosomes the mt-SSU to 3.3 Å, and the head of the mt-SSU amounts of mitochondrially encoded proteins is the enlargement of the mitoribosomal pro- to 3.5 Å (fig. S2B). These maps were used to im- (13), although a second proposed role for Cox24 teome together with contraction of the mt-rRNA prove the published model of the mt-LSU (4) (fig. in mitochondrial RNA processing (13) cannot (2, 3, 7). In contrast, the yeast mitoribosome S3) and to build a de novo model of the mt-SSU. be explained from our structure alone. Two re- The complete model of the yeast mitoribo- ported constituents of the yeast mitoribosome, 1 some contains two rRNA molecules (21S rRNA Rsm22 (14) and Yms2 (15), were not located. This MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. 2SciLifeLab, Stockholm University, in the mt-LSU and 15S rRNA in the mt-SSU), 73 adds to evidence that Rsm22 is an RNA methyl- SE-106 91 Stockholm, Sweden. proteins (table S2), and a single mt-tRNA bound transferase that only transiently interacts with *Corresponding author. Email: ramak@mrc-lmb.cam.ac.uk at the E site (fig. S4) that is presumably a mixture the mitoribosome (16, 17). Desai et al., Science 355, 528–531 (2017) 3 February 2017 1 of 4 R ES E A RC H | R E PO R T Most yeast mitoribosomal pro- mitoribosome, which, presumably as a teins with homologs in other ribo- Central result of fewer intersubunit contacts Head (h) somes have N- and C-terminal Protuberance due to a remodeled helix 44 (5, 6), extensions. These extensions in- mt-SSU (cp) can sample a more extensive con- crease the interconnectivity of the formational space than bacterial ribo- mt-SSU (fig. S7) but rarely show somes (3). The “ratcheted” and “rolled” conservation in length, sequence, mt-LSU conformations seen in the human mito- or structure with the extensions ribosome (5) and the hyper-rotated of the related proteins in the mam- state of the yeast mitoribosome seen malian mitoribosome (fig. S8A). by electron cryotomography (20) were Therefore, most protein extensions not observed. appear to have occurred after the An 8° rotation of the head in class mitoribosomes diverged, but re- Body B results in the formation of additional gions of conservation will help to Protuberances bridges between the head of the mt- reconstruct the evolution of the SSU and the central protuberance of mitoribosome. Body (b) the mt-LSU. In particular, it brings Overall, the preferential addi- the guanosine triphosphatase, mS29 tion of mitochondria-specific pro- mS29 (Rsm23), into contact with an expan- teins and protein extensions to the mS35 sion segment (H82-ES4) of the 21S uS5muS9m uS13m uS7m uS3m mS46 uS11m mt-SSU periphery creates a dis- uS2m mS37 mS43 rRNA (fig. S11). The location of this uS10m Downloaded from http://science.sciencemag.org/ on December 19, 2018 tinctive morphology compared with protein at the intersubunit interface other ribosomes (Fig. 1C and fig. uS14m in the mammalian mitoribosome in a S8B). The solvent-exposed side of uS19m bS1m guanosine 5′-diphosphate–bound state mS42 bS18m the mt-SSU body is dominated by mS23 mS45 mS33 (5, 6), together with assays showing bS21m two protein-rich protuberances. uS12m mS38 a higher affinity for guanosine 5′- The largest protuberance adjacent uS4m bS6m triphosphate (GTP) by the mt-SSU mS47 to the mRNA channel exit is formed mS26 uS15m over the intact monosome (21), had mS44 uS8m by a heterodimer of mS42 (Rsm26) bS16m mS41 uS17m led to suggestions that nucleotide and mS43 (Mrp1), which share hydrolysis is linked to subunit as- structural homology with iron- and sociation. However, the transitory lo- manganese-binding superoxide dis- cation of mS29 at the interface of the mutases (Fig. 2C). However, the yeast mitoribosome suggests that the metal-binding cores of each of these role of mS29 in the mitoribosome may proteins have diverged in sequence, be more complex or species dependent rendering a likely inactive hydro- than previously thought. Furthermore, phobic environment. This supports we cannot exclude the possibility that the idea that mitoribosomes have GTP is present given the ambiguous expanded their proteomes by ac- density for the nucleotide in the yeast quiring proteins from the mito- mt-SSU. chondrial matrix, with the original 180° The path taken by mRNA through functions of these proteins sub- the yeast mitoribosome can be traced sequently lost (2). by comparison with mRNA-bound An exception to this loss of func- Fig. 1. The structure of the yeast mitoribosome. (A) The overall structure structures of the bacterial ribosome tionality is mS47 (Ehd3), which of the complete yeast mitoribosome. (B) The mitoribosomal proteins of the (22, 23) (Fig. 4). The path curves forms the other protuberance of the yeast mt-SSU. (C) Protein elements of the mt-SSU colored by conservation; around helix 28 of the 15S rRNA that yeast mitoribosome and appears to elements conserved with the bacterial ribosome are in blue, elements conserved forms the neck connecting the head be a catalytically active enzyme. mS47 with the human mitoribosome are in red, and elements specific to the yeast and body of the mt-SSU in a non- is structurally similar to human 3- mitoribosome are in yellow. rRNA is colored gray. covalently closed channel. As in bac- hydroxyisobutyryl-coenzyme A (CoA) teria, the mRNA enters the channel hydrolase (HIBCH) (Fig. 2D), a mito- between the head and shoulder of chondrial protein with a putative role in valine some or act as a link between translation and the mt-SSU through a narrow entry site formed catabolism and hereditary mitochondrial disease the catabolic state of the mitochondrion. by uS3m (Var1), uS4m (Nam9), and uS5m (Mrps5). (18) but not a constituent component of the The mitoribosomal subunits are connected The constriction and the reported helicase activity human mitoribosome (5). Consistent with mS47 through a series of plastic intersubunit bridges of uS3 and uS4 in bacteria (24) are thought to being an active enzyme as well as an integral part (Fig. 3A, figs. S9 and S10, and table S3), which ensure that only unpaired mRNA enters the of the yeast mitoribosome, mS47 retains a solvent- regulate the relative movement of the subunits. channel. Although mitochondria-specific proteins exposed cavity likely capable of accommodating a Most of the bridges present in the bacterial ribo- mS35 (Rsm24) and mS45 (Mrps35) contribute to substrate and active-site residues that are either some, with the exception of bridges B1a/b and the architecture of the outer mRNA entrance, the preserved or show conservative mutations (Fig. B4, are also present in the yeast mitoribosome large-scale remodeling that occurs in the mam- 2D). In addition, recombinant yeast mS47 has (fig. S9). In addition, there are nine mitochondria- malian mitoribosome has not occurred (5). been shown to be capable of hydrolyzing 3- specific bridges in class A, of which only two are The path exposes mRNA at the intersubunit hydroxyisobutyryl-CoA, although the identity of also present in the mammalian mitoribosome interface for recognition by mt-tRNAs. At the A site, its in vivo substrate is unknown (19). At its (fig. S10). The extensive intersubunit bridges the mRNA threads through the major groove of the peripheral location, mS47 is unlikely to play an may restrain the movement of the yeast mito- upper part of helix 44. Here, the nucleotides G644, essential role in mitochondrial translation, ribosomal subunits, with the three observed con- A1584, and A1585 (the equivalent of G530, A1492, consistent with viable null mutants, but may formations related by subtle movements only (Fig. and A1493 in bacteria) and a loop of uS12m (Mrps12) benefit from colocalization with the mitoribo- 3, B and C). This contrasts with the mammalian form the decoding center of the mt-SSU. The Desai et al., Science 355, 528–531 (2017) 3 February 2017 2 of 4 R ES E A RC H | R E PO R T Asn mS43 h F121 mS42 F177 F177 uS3m E124 E180 E124 mS42 uS3m SOD2 E172 PDB: mS47 cavity 3BPT mS43 Fig. 2. Features of the yeast mt-SSU. (A) The structure of the yeast mt-SSU with uS3m, mS42, mS43, and mS47 highlighted. (B) The asparagine residues of mitochondria-encoded uS3m are distributed on the protein surface. (C) mS42 mS47 and mS43 [related to (A) by a 90° rotation around the y axis] form a heterodimer that structurally resembles a yeast mitochondrial superoxide dismutase dimer (Protein Data Bank ID 3LSU). (D) mS47 [related to (A) by an approximate 180° rotation around the x axis] is a probable enzyme with a large cavity and catalytic residues conserved with human b b-hydroxyisobutyryl-CoA hydrolase (Protein Data Bank ID 3BPT). Single-letter abbreviations for the amino acid residues are as follows: E, Glu; and F, Phe. mt-SSU mt-LSU 8° Downloaded from http://science.sciencemag.org/ on December 19, 2018 mB9 h mB7 mB7 B1c B1c mB9 mB10R mt-SSU mB8 Class A mB10R mB8 h CP mt-LSU mt-LSU B2b B7a mB10 B7b B2b mB10 B2a mB2 B2a Shoulder mt-SSU mt-SSU mB3 Class B Class B b 1.5° mB3 mB11 B3 0 RMSD (Å) 12 B2c mB12 B3 B8a mB14 B5 mB13 B5 B8a 1.5° mB13 B6 mB14 mB15 B6 mB16 mB16 mB15 mt-SSU b Class A Fig. 3. Bridges and mitoribosomal dynamics. (A) Intersubunit interfaces with residues that contribute to bridges highlighted. Bridges also present in the bac- terial ribosome are in blue; mitoribosome-specific bridges conserved in the human mitoribosome are in red; and yeast-specific bridges are in yellow. Residues that mt-SSU mt-SSU form additional bridges in class B are shown in teal. (B) Class B is related to class Class C Class C A by a small rotation of the body and an 8° rotation of the head. The body atoms and head vectors (right) are colored by root-mean-square displacement (RMSD) 2.5° from their positions in class A. (C) Class C is related to class A by small rotations of the body and head. similarity of this region with the decoding centers of mechanisms of initiating translation is a wide V- The density contacts both walls of the mRNA other ribosomes indicates a conserved mechanism shaped canyon at the mRNA channel exit (Fig. 4). exit canyon: bS1m (Mrp51), bS6m (Mrp17), and of decoding. There appears to be no equivalent to The canyon is flanked on one side by the mS42- mS43 (Mrp1) on one side and uS15m (Mrps28), the P-site finger that extends from the central mS43 (Rsm26-Mrp1) heterodimer protuberance uS17m (Mrps17), and mS26 (Pet123) on the other. protuberance to contact both and A- and P-site and on the other by a series of extensions to a bS1m (Mrp51) has been shown to functionally tRNAs in the mammalian mitoribosome (5, 6). number of ribosomal proteins, including bS6m interact with the 5′ UTRs of yeast mt-mRNAs In contrast to the conserved early and inter- (Mrp17), uS15m (Mrps28), uS17m (Mrps17), bS18m (26), fulfilling a role similar to that of bS1 in mediate parts of the mRNA channel, the channel (Rsm18), and bS21m (Mrp21). mRNA occupying bacteria (27), whereas bS6m (Mrp17) (28), mS26 exit shows considerable remodeling adjacent to this canyon would help to explain ribosome- (Pet123) (29), and mS43 (Mrp1) (30) have all the E site. In bacteria, this region includes the 3′ profiling data (25) that have shown that the yeast been shown to functionally interact with Pet122, tail of the 16S rRNA that facilitates start-codon mitoribosome protects longer mRNA stretches a translational activator for cytochrome c oxi- selection during translational initiation by base during active translation than cytosolic ribosomes dase subunit III. pairing with the Shine-Dalgarno sequence of the (~38 nucleotides compared to 28). In yeast, translational activators provide a mRNA 5′ UTR. As yeast mitochondrial tran- In a subset of our particles (~60%), additional physical link between the 5′ UTRs of mitochon- scripts lack Shine-Dalgarno sequences, the 3′ density could be observed above the mRNA exit drial transcripts, the mitoribosome, and the inner end of the 15S rRNA is not constrained to a canyon (Fig. 4A). Similarly placed density was mitochondrial membrane and are necessary for position at the mRNA channel exit and instead also observed by subtomogram averaging of yeast the translation of most, if not all, yeast mito- extends into the mt-SSU body, where it is se- mitoribosomes in situ (20). Despite focused- chondrial mRNAs (2, 8). Translational activa- questered by mitoribosomal proteins. A second classification approaches (fig. S12), this density tors have been proposed to compensate for possible adaptation to the absence of Shine- could not be resolved further, likely owing to the absence of a Shine-Dalgarno sequence by Dalgarno sequences and the need for alternative compositional and conformational heterogeneity. aligning the mitoribosome on the mRNA and Desai et al., Science 355, 528–531 (2017) 3 February 2017 3 of 4 R ES E A RC H | R E PO R T Unidentified density uS15m bS1m mS26 bS6m mRNA mS43 uS17m bS21m path mS23 bS18m 40˚ b uS7m b h h Fig. 4. The extended mRNA channel exit. (A) View from the mt-LSU, showing the path of the mRNA channel (red line with arrow) around the neck of the yeast mt-SSU. Additional density is located above a canyon at the mRNA channel exit. The boxed section is shown in (B) and (C). (B) The proteins that form the canyon. (C) The canyon walls are formed predominantly by mitoribosome-specific protein elements (colored red and yellow as in Fig. 1C). defining the start codon. Furthermore, transla- 7. E. O. van der Sluis et al., Genome Biol. Evol. 7, 1235–1251 26. N. S. Green-Willms, T. D. Fox, M. C. Costanzo, Mol. Cell. Biol. tional activators are specific to individual tran- (2015). 18, 1826–1834 (1998). 8. J. M. Herrmann, M. W. Woellhaf, N. Bonnefoy, Biochim. 27. E. Hajnsdorf, I. V. Boni, Biochimie 94, 1544–1553 (2012). scripts and regulate translation beyond initiation 28. P. Haffter, T. D. Fox, Mol. Gen. Genet. 235, 64–73 (1992). Biophys. Acta 1833, 286–294 (2013). by establishing tailored microenvironments (2). 9. N. 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Email: ramak@mrc-lmb.cam.ac.uk Published 3 February 2017, Science 355, 528 (2017) DOI: 10.1126/science.aal2415 This PDF file includes: Materials and Methods Figs. S1 to S12 Tables S1 to S4 References Materials!and!Methods! ! Isolation!of!yeast!mitochondria! Yeast! mitochondria! were! harvested! as! previously! described! (4).! In! brief,! Saccharomyces, cerevisiae, were! grown! aerobically! in! YPG! media! (1%! yeast! extract,! 2%! peptone,! 3%! glycerol)! until!an!optical!density!at!600!nm!(OD600)!of!2.!The!cells!were!then!centrifuged!at!4,500!×!g,for!9! min,! the! pellet! washed! with! preFcooled! distilled! water! and! further! centrifuged! for! 15! min! at! 4,500! ×! g., Following! weighing! of! the! pellet! it! was! subsequently! resuspended! in! preFwarmed! (30°C)! DTT! buffer! (100! mM! TrisFHCl! pH! 9.3,! 10! mM! DTT)! and! left! for! 30! min! at! 30°C! in! a! shaking! incubator.! The! cells! were! pelleted! by! centrifugation! at! 3,500! ×! g, for! 10! min! at! room! temperature!and!resuspended!in!Zymolyase!buffer!(20!mM!K2HPO4FHCl!pH!7.4,!1.2!M!sorbitol)! to!an!OD600!of!0.6.!1!mg!ZymolyaseF100T!(MP!Biomedicals,!LLC)!was!added!per!gram!wet!weight! measured!earlier!and!the!solution!was!shaken!slowly!at!30°C!for!60!min.!This!was!followed!by! centrifugation!at!4,000!×!g,for!15!min!at!room!temperature.!The!pellet!was!further!resuspended! in! Zymolyase! buffer! and! centrifuged! for! a! further! 15! min! at! 4,000! ×! g.! The! pellet! was! then! resuspended!in!homogenization!buffer!(20!mM!HepesFKOH!pH!7.45,!0.6!M!sorbitol,!1!mM!EDTA)! and! lysed! with! 15! strokes! in! a! glass! homogenizer.! To! separate! the! cell! debris! and! nuclei! from! mitochondria!the!solution!was!centrifuged!at!2000!×!g,for!20!min!and!the!supernatant!collected,! followed!by!further!centrifugation!at!4500!×!g,for!20!min.!Again!the!supernatant!was!collected.! Centrifugation! at! 13,000! ×! g, for! 25! min! allowed! crude! collection! of! the! mitochondrial! pellet.! Crude!mitochondria!were!further!purified!on!15F60%!step!sucrose!gradient!in!SEM!buffer!(250! mM!sucrose,!20!mM!HepesFKOH!pH!7.5,!1!mM!EDTA)!by!ultracentrifugation!at!141,000!x!g!for!1! hour.!Mitochondrial!samples!were!pooled,!flash!frozen!using!liquid!nitrogen!and!stored!at!F80°C.!! ! Purification!of!yeast!mitoribosomes! 3!volumes!of!Lysis!buffer!(25!mM!HepesFKOH!pH!7.5,!100!mM!KCl,!25!mM!MgOAc,!1.7%!Triton! XF100,! 2! mM! DTT)! supplemented! with! 0.0075%! Cardiolipin! and! 100! µg/ml! chloramphenicol! were!added!to!mitochondria!purified!from!the!sucrose!gradient!and!incubated!for!10!min!on!ice.!! Centrifugation! at! 30,000! ×! g, for! 20! min! separated! out! the! membrane! fraction! and! the! supernatant!was!loaded!on!a!1!M!sucrose!cushion!in!buffer:!20!mM!HepesF!KOH!pH!7.5,!100!mM! KCl,! 20! mM! MgOAc,! 1%! Triton! XF100,! 2! mM! DTT,! 0.0075%! cardiolipin,! 0.05%! DDM! and! 50! µg/ml! chloramphenicol.! After! centrifugation! for! 4! hours,! the! pellet! was! resuspended! in! the! above!buffer!without!Triton!XF100!and!loaded!on!a!15%F30%!sucrose!gradient!and!run!for!16!h! at! 80,000! ×! g.! Mitoribosome! fractions! were! collected! and! the! sucrose! removed! by! passing! the! sample! through! a! 15! ml! concentrator! (Vivaspin)! with! a! 30! kDa! molecular! weight! cutoff! and! ! replenishing! with! the! same! buffer! as! above! but! without! Triton! XF100! and! with! 0.001%! cardiolipin.!! ! Grid!preparation! 3!µl!aliquots!of!purified!yeast!mitoribosomes!at!a!concentration!of!~97!nM!(0.29!mg/ml)!were! applied!onto!30!s!glowFdischarged!holey!carbon!grids!(Quantifoil!R2/2)!coated!with!homeFmade! continuous! carbon! (~50! Å! thick)! prior! to! being! blotted! for! 3.5! s! using! the! FEI! Vitrobot.! The! conditions!were!set!to!100%!ambient!humidity!and!4°C!and!the!grids!were!flash!frozen!in!liquid! ethane!prior!to!transfer!into!liquid!nitrogen!for!storage.! ! Electron!microscopy! The!grids!were!loaded!onto!an!FEI!Titan!Krios!electron!microscope!operated!at!an!accelerating! voltage!of!300!kV.!FEI’s!automated!single!particle!acquisition!software!(EPU)!was!used!to!collect! the!images,!which!were!recorded!on!a!backFthinned!FEI!Falcon!II!detector!at!a!magnification!of! 104,478! ×! (resulting! in! a! pixel! size! of! 1.34! Å).! 17! movie! frames! were! collected! for! each! 1! s! exposure.! Defocus! values! were! set! from! F1! to! F3.3! µm.! Data! were! collected! in! a! single! session! over!a!48!h!period.!! ! Image!processing! The! movie! frames! of! each! image! were! aligned! using! MotionCorr! (31)! prior! to! estimating! contrast! transfer! function! (CTF)! parameters! using! CTFFIND3! (32).! After! manual! inspection! of! the! images! and! their! corresponding! Fourier! transforms! a! total! of! 2,525! micrographs! were! retained.! 468,858! particles! were! selected! from! these! micrographs! using! semiFautomated! particle!picking!in!EMAN!2!(33).!All!subsequent!image!processing!was!performed!using!RELION! 1.4! (34).! ReferenceFfree! twoFdimensional! (2D)! classification! was! used! to! discard! 127,588! particles.! The! remaining! 341,270! particles! were! refined! using! a! 60! Å! lowFpass! filtered! map! of! the!unmasked!yeast!mitoribosome!(4)!as!an!initial!reference.!These!particles!were!subsequently! corrected! for! beamFinduced! particle! motion! using! “particle! polishing”! in! RELION.! The! nominal! resolution! after! refinement! of! these! 341,270! particles! (3.3! Å),! and! of! all! other! steps,! was! estimated!during!postFprocessing!using!the!FourierFshellFcorrelation!(FSC)!0.143!criterion!(35).! Binary! masks! created! for! postFprocessing,! refinement! and! classification! were! generated! in! RELION!with!a!soft!edge!applied.!HighFresolution!noise!substitution!was!used!to!correct!for!the! effects! of! applying! a! mask! during! the! FSC! calculations! (36).! Before! visualization,! density! maps! were!corrected!for!the!modulation!transfer!function!of!the!Falcon!II!detector!and!sharpened!by! applying!a!negative!BFfactor!that!was!estimated!using!automated!procedures!(35).! ! ! The! polished! particles! were! then! refined! with! a! mask! applied! over! the! mtFSSU! before! a! first! round! of! 3D! classification! with! the! mask! maintained.! This! classification! step! was! performed! without!further!refinement!of!the!assigned!angles,!which!allowed!the!rapid!isolation!of!classes!in! which!the!mtFSSU!adopts!different!orientations.!These!classes!were!individually!refined!before!a! second!round!of!3D!classification,!again!without!alignment!steps.!Three!wellFpopulated!classes! were! identified! (classes! A–C,! designated! on! the! basis! of! the! number! of! particles! in! each! class).! The!best!monosome!class!(class!A)!resolved!to!3.3!Å.!The!remaining!two!classes!resolved!to!3.7! Å!and!5.0!Å.! ! However,! even! in! class! A! regions! of! the! map! were! insufficiently! resolved! to! allow! for! accurate! model!building.!To!improve!local!map!density,!we!generated!masks!for!the!mtFLSU,!the!body!of! the!mtFSSU!and!the!head!of!the!mtFSSU!and!applied!these!to!separate!3D!refinements.!The!masks! of! the! mtFSSU! body! and! head! were! applied! to! 264,961! particles! after! the! first! round! of! classification,! while! the! mask! of! the! mtFLSU! was! applied! to! 221,875! particles! after! the! second! round! of! classification.! The! mtFSSU! body! had! an! additional! round! of! 3D! classification! and! 3D! refinement.! The! resulting! maps! showed! improved! resolution! and! local! map! quality! (Fig.! S2,! B! and!D).! ! Additionally,! focused! classification! with! signal! subtraction! (FCwSS)! (37)! was! performed! on! a! region! of! unidentified! density! above! the! mRNA! exit! channel! (Fig.! S12).! Starting! with! 264,961! particles! aligned! with! a! mask! over! the! mtFSSU! body,! multiple! rounds! of! FCwSS! were! used! to! isolate!a!single!class!of!153,300!particles!in!which!the!density!was!best!resolved.!! ! Model!building! The!masked!maps!were!used!for!model!building!to!take!advantage!of!the!better!local!resolution.! GaussianFfiltered! maps! generated! using! Chimera! (38)! were! used! to! build! models! in! regions! of! the!map!with!poor!density.!! ! Initially,!models!of!the!small!subunits!of!the!E.,coli,ribosome!(Protein!Data!Bank!ID!5IQR)!(39)! and! the! human! mitoribosome! (Protein! Data! Bank! ID! 3J9M)! (5)! were! fitted! to! the! map! of! the! yeast! mtFSSU! using! the! “fit! in! map”! function! of! Chimera! (38).! Each! ribosomal! protein! in! the! template! models! was! then! extracted! with! those! lacking! density! removed.! All! subsequent! modeling!was!done!in!Coot!(40)!.!Proteins!with!homologs!in!the!yeast!mitoribosome!were!rigidF body! fitted! to! the! density! and! modified! to! the! sequence! and! numbering! of! the! related! yeast! mitoribosomal!protein!using!sequence!alignments!from!ClustalOmega!(41).!The!model!was!then! fitted! to! the! density! using! realFspace! refinement.! Extensions! and! insertions! were! modeled! de, novo! in! Coot.! During! model! building! torsion,! planarFpeptide,! transFpeptide! and! Ramachandran! ! restraints!were!applied.!Helix!restraints!were!applied!during!realFspace!refinement!of αFhelices.! TransFpeptide!restraints!were!removed!to!model!cisFproline!residues!for!which!there!were!clear! density.!!! ! Sections!of!the!map!without!known!homologs!were!interpreted!with!poly(alanine)!models!that! were! then! searched! against! protein! databases! using! PDBeFold! (42),! DALI! (43)! and! Backphyre! (44)! to! help! identify! yeastFspecific! mitoribosomal! proteins.! Sequences! estimated! from! the! density!were!searched!against!a!list!of!potential!yeast!mitoribosomal!proteins!identified!by!mass! spectrometry!or!a!nonFredundant!set!of!protein!sequences!using!protein!BLAST!(45).!! ! Mitoribosomal! proteins! of! the! yeast! mtFSSU! were! named! according! to! the! recommended! nomenclature,! as! well! as! mtFLSU! protein! Mhr1! now! renamed! to! mL67! (3).! The! yeast! mitochondriaFspecific!proteins!were!numbered!starting!with!the!lowest!number!without!a!prior! assignment!(mS41)!with!the!proteins!ordered!by!increasing!molecular!weight.!! ! The!15S!rRNA!(GenBank!ID:!KP263414)!was!built!using!the!model!of!E.,coli!16S!rRNA!(Protein! Data! Bank! ID! 5IQR)! (39)! as! a! template.! Using! information! from! an! alignment! of! the! two! sequences! from! ClustalOmega! (41)! and! their! respective! secondary! structure! diagrams! (46),! conserved! sections! were! extracted! from! the! globally! fitted! 16S! rRNA! structure! and! rigidFbody! fitted! to! the! density! to! overcome! local! differences.! The! sequence! and! numbering! of! the! bases! were!then!altered!to!that!of!S.,cerevisiae!15S!rRNA.!Where!necessary,!these!conserved!sections! were! connected! by! de,novo! modeling.! Each! nucleotide! was! inspected! and! fitted! to! the! density! using! realFspace! refinement,! often! using! the! “sphere”! refinement! tool! Coot! to! include! all! nucleotides!in!the!surrounding!environment!including!the!baseFpaired!partner.! To!improve!the!previously!published!model!of!the!yeast!mtFLSU!(Protein!Data!Bank!ID!3J6B)!(4)! we! first! docked! the! model! to! the! map! in! Chimera! and! then! optimized! the! fit! using! realFspace! refinement! in! Coot.! Particular! emphasis! was! applied! to! correcting! Ramachandran! outliers! and! regions!of!the!model!at!the!mitoribosome!periphery.!Previously!unbuilt!sections!of!the!proteins! were! modeled! where! the! density! permitted.! The! improved! map! quality! allowed! two! short! regions! of! the! 21S! rRNA! with! registry! errors! to! be! identified.! Nucleotides! 535–557! were! renumbered!to!531–553!and!nucleotides!3005–3039!were!renumbered!to!3002–3035,!with!the! corresponding!sequences!changed.!! ! Model!refinement!and!validation! Models! for! each! of! the! domains! (mtFSSU! body,! mtFSSU! head! and! mtFLSU)! were! first! refined! against! their! respective! masked! maps! to! take! advantage! of! the! higher! resolution! information.! ! The! refined! models! were! then! combined! and! further! refined! against! each! of! the! three! yeast! mitoribosomal!classes.!! ! Prior! to! each! refinement,! secondary! structure! restraints! for! the! mitoribosomal! proteins! were! derived! from! the! model! using! ProSMART! (47).! Basepair,! stacking! and! sugarFpucker! restraints! were! obtained! for! the! rRNA! using! LIBG! (48).! These! external! restraints! were! applied! during! reciprocalFspace! refinement! in! REFMAC! v5.8! optimized! for! cryoFEM! maps! (48).! Tighter! restraints! were! applied! during! refinement! against! the! lower! resolution! maps! to! maintain! the! stereochemistry!obtained!from!the!refinements!with!the!higher!resolution!masked!maps.!During! refinement,! the! fit! of! the! model! to! the! map! density! was! quantified! using! FSCaverage! (48).! Information! beyond! the! nominal! map! resolution! estimated! by! the! FSC=0.143! criterion! was! excluded! during! refinement! and! preserved! for! validation.! The! final! modelFtoFmap! fit! was! evaluated! using! FSCaverage! and! CRef! (35).! CRef! is! a! measure! of! the! resolution! when! the! FSC! between!the!refined!model!and!map!is!0.5.!! ! PostFrefinement!model!statistics!were!obtained!from!REFMAC!and!MolProbity!(49)!and!given!in! table!S1.!CrossFvalidation!against!overfitting!was!performed!as!described!(4,!48).! ! Figure!preparation!! Figures! featuring! cryoFEM! maps! were! generated! using! Chimera! (38).! Maps! colored! by! local! resolution! were! generated! using! estimations! of! resolution! by! ResMap! (50).! Figures! featuring! only!models!were!generated!using!PyMOL!(51).!!! ! The! secondary! structure! diagram! for! S., cerevisiae! 15S! rRNA! was! created! by! modifying! the! diagram!from!the!Comparative!RNA!Website!(46)!with!baseFpair!information!extracted!from!the! final!model!using!DSSR!(52).!rRNA!helices!were!labeled!according!to!the!equivalent!helices!in,E., coli!16S!rRNA.!! ! The! proteinFprotein! interaction! network! of! the! yeast! mtFSSU! was! mapped! in! twoFdimensions! using! CytoScape! (53).! Molecular! weights! from! UniProt! (54)! were! used! to! represent! the! nodal! size! and! the! edge! thickness! represents! the! extent! of! the! protein–protein! interface! calculated! from!our!model!using!PDBePISA!(55).!! ! ! ! Table!S1.!Data!and!model!statistics.! ! Class!A! Class!B! Class!C! Data!Collection! ! ! ! ! Particles! 141,795! 55,448! 24,632! ! Pixel!size!(Å)! 1.34! 1.34! 1.34! ! Defocus!mean!(µm)! 2.4! 2.5! 2.6! ! Defocus!range!(µm)! 1.3–4.8! 1.3–4.8! 1.3–4.8! ! Voltage!(kV)! 300! 300! 300! ! Electron!dose!(e·Å−2)! 23.5! 23.5! 23.5! Model!composition! ! ! ! ! NonFhydrogen!atoms! 201,471! 201,471! 201,471! ! Protein!residues! 13,711! 13,711! 13,711! ! RNA!bases! 4,286! 4,286! 4,286! ! Ligands!(Zn2+/Mg2+)! 2/301! 2/301! 2/301! Refinement! ! ! ! ! Resolution!(Å)! 3.25! 3.75! 4.97! ! Map! sharpening! BFfactor! F72.0! F86.6! F138.0! (Å2)! ! Average!B!factor!(Å2)! 74.9! 87.0! 111.0! ! FSCaverage! 0.80! 0.77! 0.72! ! CRef!(Å)! 3.37! 3.98! 5.78! Rms!deviations! ! ! ! ! Bond!lengths!(Å)! 0.005! 0.005! 0.005! ! Bond!angles!(°)! 1.03! 1.02! 1.02! Validation!(proteins)! ! ! ! ! MolProbity!score! 2.0!! 2.1!! 2.0!! (100th!percentile)! (100th!percentile)! (100th!percentile)! ! Clashscore,!all!atoms! 2.4!! 3.1!! 2.7!! (100th!percentile)! (100th!percentile)! (100th!percentile)! ! Favored!rotamers!(%)! 84.8! 84.9! 84.7! ! Poor!rotamers!(%)! 6.0! 6.0! 6.0! Ramachandran!plot! ! ! ! ! Favored!(%)! 94.3! 94.4! 94.3! ! Outliers!(%)! 0.4! 0.4! 0.4! Validation!(RNA)! ! ! ! ! Correct!sugar!puckers!(%)! 95.3! 95.3! 95.3! ! Good! backbone! 74.5! 74.7! 74.6! conformations!(%)! ! Table& S2.& Mitoribosomal& proteins& (MRPs)& of& the& yeast& mt:SSU.!A!similar!table!for!the!MRPs!of!the!yeast!mt4LSU!can!be!found!in!(4).!The!MRPs!are! colored!by!conservation!with!the!bacterial!ribosome!(blue)!and!the!human!mitoribosome!(red)!or!exclusivity!to!the!yeast!mitoribosome!(yellow).! ! MRP& Alias& UniProt& Chain& Mature& MW& Modeled&residues& Notes& ID& ID& protein& (Da)& residue& range& bS1m! Mrp51! Q02950! AA! 14344! 39,445! 24109,! 1954233,! Extensively!remodeled!compared!to!its!bacterial! 2444299,! and!human!mitochondrial!counterpart.!! uS2m! Mrp4! P32902! BB! 264394! 44,162! 1284393! ! uS3m! Var1! P02381! CC! 14398! 47,123! 30468,!784158,!1664 Encoded!by!the!yeast!mitochondrial!genome.!! 280,!2954398! uS4m! Nam9! P27929! DD! 354486! 56,356! 2480,! 884192,! 3844 ! 486! uS5m! Mrps5! P33759! EE! 144307! 34,883! 144113,!1194306! ! bS6m! Mrp17! P28778! FF! 14131! 15,021! 14125! ! uS7m! Rsm7! P47150! GG! 274247! 27,816! 874247! ! uS8m! Mrps8! Q03799! HH! 14155! 17,471! 24155! ! uS9m! Mrps9! P38120! II! 114278! 31,925! 35471,!814134,!1444 ! 278! uS10m! Rsm10! Q03201! JJ! 154203! 23,424! 164201! ! uS11m! Mrps18! P42847! KK! 604217! 24,563! 70499,!1064217! ! uS12m! Mrps12! P53732! LL! 214153! 16,917! 294152! ! uS13m! Sws2! P53937! MM! 14143! 16,089! 24121! ! uS14m! Mrp2! P10663! NN! 14115! 13,538! 14115! ! uS15m! Mrps28! P21771! OO! 344286! 33,057! 344112,!1284286! ! bS16m! Mrps16! Q02608! PP! 14121! 13,639! 24106,!1104120! ! uS17m! Mrps17! Q03246! QQ! 14237! 27,635! 24117,1244135,! ! 1404150,! 1594208,! 2184232! bS18m! Rsm18! P40033! RR! 14138! 15,835! 40472,!814138! ! ! uS19m! Rsm19! P53733! SS! 1491! 10,275! 9488! ! bS21m! Mrp21! P38175! TT! 184177! 20,395! 864177! ! ! mS23! Rsm25! P40496! UU! 14264! 30,513! 14233! ! mS26! Pet123! P17558! VV! 14318! 35,998! 24234! ! mS29! Rsm23! Q01163! WW! 154450! 50,867! 504450! ! mS33! Rsm27! P53305! XX! 14110! 12,393! 1496! ! mS35! Rsm24! Q03976! YY! 314319! 37,393! 474119,!1244319! ! mS37! Mrp10! O75012! ZZ! 2495! 10,691! 5470,!75495! Although! yeast! mS37! shares! some! similarity! to! human! mS37,! it! adopts! a! different! spatial! orientation.! Yeast! mS37! contains! two! pairs! of! cysteine! residues! that! may! form! disulfide! linkages! in$ vivo! (56),! but! are! not! modeled! as! doing! so! here! due! to! insufficient! density! for! a! disulfide!bond.!! mS38! Cox24! P32344! 11! 14111! 12,772! 784111! Not! previously! identified! as! a! mitoribosomal! protein!in!yeast.!! ! mS41! Fyv4! P38783! 22! 284130! 15,292! 304128! Sterile!alpha!motif!domain! mS42! Rsm26! P47141! 33! 14266! 30,224! 84100,!1124262! Forms!a!heterodimer!with!mS43.!Homologous!to! Fe/Mn!superoxide!dismutases.! mS43! Mrp1! P10662! 44! 144421! 36,729! 184176,! 1904230,! Forms!a!heterodimer!with!mS42.!Homologous!to! 2444313! Fe/Mn!superoxide!dismutases.! mS44! Mrp13! P12686! 55! 384339! 38,988! 42478,!1154136! ! mS45! Mrps35! P53292! 66! 274345! 39,575! 274276,!2914345! ! mS46! Rsm28! Q03430! 77! 14359! 41,216! 1974361! Occupies! a! similar! position! to! human! mS31! but! any! structural! resemblance! appears! to! be! a! result! of! constraints! of! the! environment! rather! than!a!shared!ancestry.! mS47! Ehd3! P28817! 88! 364500! 56,288! 364449,!4554492! Probable! active! enzyme.! Structural! similar! to! human! 34hydroxyisobutyryl4CoA! hydrolase! (HIBCH,!PDB!entry!code!3BPT).! ! Table&S3.&Intersubunit1bridge&composition.&The!bridges!are!colored!by!conservation!with!the! bacterial! ribosome! (blue)! and! the! human! mitoribosome! (red)! or! exclusivity! to! the! yeast! mitoribosome!(yellow)!and!the!subset!of!these!found!in!class!B!only!(teal).!! ! Bridge& mt1SSU& mt1LSU& B1c! uS10m:!43B44,!69! bL31m:!97B98,!101,!! h42:!1379B1380! 104B105! B2a! h44:!1474B1477,!1586B1599! H69:!1813B1817,!1819B1820! h45:!1609! h24:!858! B2b! h24:!848B850! H68:!1744B1745! 21S:!1827B1829! B2c! h24:!835B838! H66:!1698B1701! h27:!964B965! H67:!1738B1741! B3! h44:!1575B1577! H71:!1846B1852,!1859B1861! 1487B1490! uL14m:!49B50,!65! B5! h44:!1496B1497! H62:!1656B1657! B6! h44:!1556B1557! bL19m:!155! B7a! h23:!768! H68:!1754B1755! ! B7b! h23:!778B779! uL2m:312,!314B315! h24:!838B839! 274! B8a! h14:!343B345! uL14m:!13,!48,!110,!112B114! ! mB2! bS6m:!87B90! uL2m:!228B229,!232B233! 57B58! 247B248! mB3! mS38:!101B102! H70:!1832B1834! 105B106! H71:!1859B1860! 109B110! H62:!1643B1644! H67:!1739B1741,!1874B1877,!! ! mB7! mS29:!241! H82BES4:!2389B2390! mB8! uS13m:!56! mL46:!72,!74! mB9! uS13m:!70! uL5:!253B254! mB10R! uS19m:!67! uL5:!218B219! uS13m:!84! ! mB10! uS19m:!63B67! uL5:!218B219,!241! uS13m:!84! mB11! uS17m:!105B111! uL2m:!61B88! uS15m:!77B84,!145! bS6m:!35B36,!74B83! h22:735B736! mB12! uS15m:!268,!272,!275,!279,!282B283! H42:!613B614! H62:!1639B1640,!1645B1648! mB13! uS13m:!34,!37B38,!41! H62:!1657B1658! mB14! h14:315! bL19m:!166! mB15! mS44:!72,!75B76! bL19m:!135B138! mB16! h44:!1510B1511! H101BES:!3172! ! ! ! Table& S4.& Mass& spectrometry& analysis.& Purified& yeast!mitoribosomes!were!analyzed!by!mass! spectrometry! (4)! revealing! the! presence! of! a! number! of! translational! activators! and! mitochondrial!proteins!of!various!functions!(others).!& ! mt1LSU& & mt1SSU& & Translational& ! Others! Activators& uL1m! uL30m! ! bS1m! ! Aep1! ! Ach1! Lpd1! uL2m! bL31m! ! uS2m! ! Aep2! ! Aco1! Lsc2! uL3m! bL32m! ! uS3m! ! Atp25! ! Ald4! Lsp1! uL4m! bL33m! ! uS4m! ! Cbp1! ! Atp1! Mdh1! uL5m! bL34m! ! uS5m! ! Cbs2! ! Atp16! Mic10! uL6m! bL36m! ! bS6m! ! Mam33! ! Atp2! Mir1! bL9m! mL38! ! uS7m! ! Ssc1! ! Atp3! Mss116! uL10m! mL40! ! uS8m! ! !! ! Atp4! Ndi1! uL11m! mL41! ! uS9m! ! !! ! Atp5! Om45! bL12m! mL43! ! uS10m! ! !! ! Atp6! Pda1! uL13m! mL44! ! uS11m! ! !! ! Atp7! Pdb1! uL14m! mL46! ! uS13m! ! !! ! Cat2! Pdx1! uL15m! mL49! ! uS14m! ! !! ! Cit1! Pet9! uL16m! mL50! ! uS15m! ! !! ! Cor1! Phb1! uL17m! mL53! ! bS16m! ! !! ! Cox2! Phb2! bL19m! mL54! ! uS17m! ! !! ! Cox5! Por1! bL21m! mL57! ! bS18m! ! !! ! Fum1! Pth4! uL22m! mL58! ! bS21m! ! !! ! Ggc1! Qcr2! uL23m! mL59! ! mS23m! ! !! ! Gut2! Qcr7! uL24m! mL60! ! mS26! ! !! ! Hsp60! Rip1! bL27m! mL61! ! mS29! ! !! ! Idh2! Sdh1! bL28m! MHR1! ! mS33! ! !! ! Ilv5! Sdh2! uL29m! ! ! mS35! ! !! ! Kgd1! Stb1! ! ! ! mS37! ! !! ! Kgd2! Suv3! ! ! ! mS41! ! !! ! Krt14! Tim9! ! ! ! mS42! ! !! ! Lat1! ! ! ! ! mS43! ! !! ! ! ! ! ! ! mS44! ! !! ! ! ! ! ! ! mS45! ! !! ! ! ! ! ! ! mS46! ! !! ! ! ! ! ! ! mS47! ! !! ! ! ! ! & & ! Supplementary&figures& & Fig.& S1.& Electron& microscopy& data& and& processing& work& flow.& (A)! Representative! electron! micrograph.! (B)! Gallery! of! 2D! classes! showing! different! views! of! the! yeast! mitoribosome.! (C)! Data!processing!steps.& ! & ! & & Fig.& S2.& Map& quality.& (A)! FourierBshellBcorrelation! (FSC)! curves! for! the! three! different! conformations!of!the!yeast!mitoribosome.!(B)!FSC!curves!for!the!masked!maps.!(C)!The!map!for! each! class! colored! by! local! resolution.! (D)! The! map! for! each! masked! map! colored! by! local! resolution.! (E)! FSC! curves! of! the! fit! of! the! refined! model! to! the! final! map! (black)! for! each! conformation! of! the! yeast! mitoribosome.! The! resolution! at! FSC=0.5! (CRef)! is! indicated! with! a! dashed!line.!The!selfB!(FSCwork)!and!crossBvalidated!(FSCfree)!correlations!are!shown!in!blue!and! red,!respectively.! ! & ! & & Fig.& S3.& An& improved& map& for& the& yeast& mt1LSU.& (A)! FourierBshellBcorrelation! (FSC)! curves! comparing!the!masked!map!of!the!yeast!mtBLSU!with!our!previously!published!map!(EMDB2566)! (4).!Although!the!overall!resolutions!are!similar!(~3.2!Å,!according!to!the!FSC=0.143!criterion)! the! new! map! has! higher! correlations! at! lower! resolutions.! (B)! Comparison! of! the! final! postB processed! maps.! (C)! Examples! of! regions! that! could! be! interpreted! with! a! model! as! a! result! of! improved!map!density.!& ! ! ! ! Fig.& S4.& The& yeast& mitoribosome& co1purifies& with& an& E1site& mt1tRNA.!(A)!Slice!through!the! unfiltered!map!of!the!yeast!mitoribosome!showing!an!EBsite!mtBtRNA!bound!in!the!intersubunit! space& that! is! presumably! a! mixture! of! the! 24! mtBtRNAs! encoded! by! the! yeast! mitochondrial! genome.!The!acceptor!stem!and!elbow!of!the!mtBtRNA!contacts!the!mtBLSU,!while!the!anticodon! stemBloop!contacts!the!mtBSSU.!(B)!Sharpened!map!of!the!mtBtRNA.!SubunitBmasked!maps!were! used!to!model!the!acceptor!and!anticodon!arms!of!an!unidentified!tRNA!into!the!map.!& ! ! ! ! Fig.& S5.& Secondary& structure& diagram& of& the& yeast& 15S& rRNA.& (A)! 15S! rRNA! colored! by! domain.! The! rRNA! expansions! specific! to! the! yeast! mitoribosome! are! highlighted! in! red.! Nucleotides! that! could! not! be! modeled! due! to! poor! density! are! shown! with! no! background! coloring.! Red! lettering! is! used! for! unmodeled! areas! specific! to! the! yeast! mitoribosome.! (B)! Secondary! structure! diagram! of! the! E.$ coli! 16S! rRNA! with! helices! not! present! in! the! yeast! mitoribosome!shown!in&blue.!! & ! ! & ! Fig.& S6.& Fit! of! the! mS38! (Cox24)! model! to! density.! mS38! was! identified! as! being! a! constituent! component!of!the!yeast!mitoribosome!from!the!density.!&! ! ! & ! & Fig.& S7.& Interactions& between& the& proteins& of& the& yeast& mt1SSU.& (A)! Yeast! mitoribosomal! proteins!with!homologs!in!the!bacterial!ribosome!are!shown!in!blue!and!those!with!homologs!in! the!human!mitoribosome!are!shown!in!red.!YeastBspecific!proteins!are!shown!in!yellow.!(B)!The! yeast! mtBSSU! visualized! as! a! proteinBprotein! network.! The! nodal! size! represents! the! relative! molecular!masses!of!the!mitoribosomal!proteins!and!the!edge!thickness!represents!the!extent!of! the!interface!between!interacting!proteins.!Interactions!that!also!occur!in!the!bacterial!ribosome! and!the!human!mitoribosome!are!shown!in!blue!and!red!respectively.!Interactions!specific!to!the! yeast! mitoribosome! are! shown! in! grey.! Edgeless! nodes! represent! proteins! that! contact! rRNA! only.!! ! ! ! ! Fig.& S8.& Mitoribosomal& protein& expansion.& (A)! The! tertiary! folds! of! each! mitoribosomal! protein! of! the! yeast! mtBSSU! colored! by! conservation.! Elements! conserved! with! the! bacterial! ribosome!are!colored!blue.!Elements!present!in!the!human!mitoribosome!but!not!the!bacterial! ribosome! are! colored! red.! YeastBspecific! elements! are! colored! yellow.! (B)! Distinct! protein! and! rRNA! elements! give! the! yeast! mitoribosome! a! distinct! architecture! compared! to! the! human! mitoribosome!and!the!bacterial!ribosome.& ! ! ! Fig.&S9.&Bridges&conserved&with&the&other&ribosomes.&Molecular!details&for!each!intersubunit! bridge! of! the! yeast! mtBSSU! that! either! occurs! in! the! bacterial! ribosome! or! in! the! human! mitoribosome.!Bridges!only!present!in!mitoribosomes!are!prefixed!with!an!“m”.!! & & ! & Fig.&S10.&Bridges&specific&to&the&yeast&mitoribosome.&Molecular!details&for!each!intersubunit! bridge!specific!to!the!yeast!mitoribosome.!Bridges!mB7,!mB8,!mB9!and!mB10R!are!only!present! in! class! B! where! an! 8°! rotation! of! the! mtBSSU! head! results! in! additional! contacts! with! the! mtB LSU.!! ! ! ! & Fig.&S11.&Comparison&of&mS29&positions&in&yeast&and&human&mitoribosomes.&(A)!Position!of! mS29!in!the!yeast!mitoribosome!class!A,!with!a!close!up!of!the!boxed!section.!The!boxed!sections! in!panels!B!and!C!represent!equivalent!areas!in!the!yeast!mitoribosome!class!B!and!the!human! mitoribosome!respectively.!(B)!Owing!to!an!8°!rotation!of!the!mtBSSU!head,!mS29!contacts!H82B ES4!of!the!mtBLSU!rRNA!in!the!yeast!mitoribosome!class!B!(bridge!mB7).!(C)!Equivalent!position! of!mS29!in!the!human!mitoribosome,!where!the!mtBLSU!contact!is!mediated!by!mL46!and!mL48.! ! ! ! ! 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