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1 doi: /nature12890 Supplementary Table 1 Summary of protein components in the 39S subunit model. MW, molecular weight; aa amino acids; RP, ribosomal protein. Protein* MRP size MW (kda) Sequence accession code Chain ID Modeled residues Coverage (%) Modeling template (PDB ID, chain ID) homolog* RP size Comments with homologs in bacterial ribosomes MRPL AK L1 229 MRPL NP_ D V2D (D) L2 276 MRPL XP_ E V2D (E) L3 206 MRPL XP_ F V2D (F) L4 210 residues and MRPL XP_ I V2D (I) L9 148 MRPL XP_ L MRPL XP_ L MRPL AK L7/L MRPL NP_ N V2D (N) L MRPL XP_ O V2D (O) L MRPL NP_ P V2D (P) L MRPL NP_ Q V2D (Q) L MRPL NP_ R V2D (R) L MRPL XP_ S V2D (S) L MRPL XP_ T V2D (T) L MRPL XP_ U V2D (U) L MRPL XP_ V V2D (V) L MRPL AK W V2D (W) L MRPL AK X V2D (X) L23 96 MRPL NP_ Y V2D (Y) L MRPL XP_ V2D (0) L27 85 MRPL XP_ V2D (1) L28 98 MRPL XP_ R73 (A) L29 72 MRPL XP_ V2D (3) L30 60 MRPL XP_ Zn V2D (5) L32 60 MRPL XP_ V2D (6) L33 54 MRPL AW V2D (7) L34 49 MRPL XP_ V2D (8) L35 65 MRPL XP_ MRPL AK Zn 500 without homologs in bacterial ribosomes residues 9-21 residues residues residues residues residues and residues not modelled Zn 2+ binding; residues V2D (9) L36 37 Zn 2+ binding MRPL XP_ b QYQ (J) PEBP-like fold MRPL XM_ c QF6 (A) trna synthetase domain like residues
2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 1 (continued) Protein* MRP size MW (kda) Sequence accession code Chain ID Modeled residues Coverage (%) Modeling template (PDB ID, chain ID) homolog* RP size Comments without homologs in bacterial ribosomes MRPL NP_ MRPL AW MRPL AY MRPL XP_ thioredoxin like MRPL NP_ h O0W (B) RNase III domainlike residues UNK MRPL AK i CW9 (G) cystatin-like MRPL XP_ nudix hydrolase MRPL XP_ ferredoxin-like MRPL NP_ l XZN (Z) eif1-like MRPL XP_ ACP-like MRPL I3LJG7 (Uniprot) MRPL NP_ o residues MRPL XM_ thioredoxin-like MRPL XP_ MRPL NP_ MRPL NP_ lactamase/ transpeptidaselike MRPL57 (MRP63) MRPL58 (ICT1) MRPL59 (CRIF1) AK NP_ u J26 (A) XP_ v, w z x 5 helices (91 aa) each 21 helices (426 aa) of template 1S3A (A) homeodomainlike peptidyl-trna hydrolase residues not modeled unassigned helices of dimeric or pseudo dimeric protein fold unassigned helices unassigned thioredoxin fold * Nomenclature according to the ribosomal protein gene database 1. Length, molecular weight and sequence coverage are calculated based on the full-length protein sequence (i.e. including putative mitochondrial targeting peptides). Unassigned residues were modeled as poly-serine and deposited as UNK. Fold predicted by the Phyre2 protein fold recognition server
3 RESEARCH Supplementary Table 2 Overview of statistically significant hits for interprotein chemical crosslinks of 39S subunit proteins obtained from CX-MS experiments using S. scrofa 39S subunits and B. taurus 55S mitoribosomes. 1 2 Organism MRPL11 K13 MRPL53 K14 S. scrofa MRPL11 K12 MRPL53 K14 S. scrofa MRPL22 K54 MRPL24 K100 S. scrofa MRPL38 K274 MRPL52 K111 S. scrofa MRPL28 K12 MRPL37 K40 S. scrofa MRPL38 K85 MRPL52 K105 S. scrofa MRPL3 K330 MRPL19 K97 S. scrofa MRPL9 K81 MRPL28 K51 S. scrofa MRPL22 K54 MRPL24 K100 S. scrofa MRPL1 K297 CRIF1 K113 S. scrofa ICT1 K197 MRPL18 K114 S. scrofa MRPL38 K305 MRPL52 K105 S. scrofa MRPL38 K85 MRPL52 K105 S. scrofa MRPL20 K26 MRPL44 K43 S. scrofa MRPL9 K61 MRPL28 K51 S. scrofa MRPL38 K85 MRPL52 K98 S. scrofa ICT1 K93 MRPL49 K158 S. scrofa ICT1 K165 MRPL19 K227 S. scrofa MRPL12 K142 MRPL53 K14 S. scrofa MRPL11-1/-2 K51/K25* MRPL12 K183 S. scrofa MRPL46 K100 MRPL40 K97 S. scrofa MRPL30 K44 MRPL16 K238 S. scrofa MRPL24 K32 MRPL45 K62 S. scrofa MRPL45 K62 MRPL24 K100 S. scrofa MRPL23 K115 MRPL45 K54 S. scrofa MRPL12 K138 MRPL53 K14 S. scrofa MRPL38 K85 MRPL52 K105 S. scrofa MRPL53 K105 MRPL11 K13 S. scrofa MRPL38 K305 MRPL52 K107 S. scrofa MRPL10 K132 MRPL53 K25 S. scrofa MRPL47 K136 MRPL9 K81 S. scrofa MRPL38 K305 MRPL52 K111 S. scrofa MRPL15 K249 ICT1 K66 S. scrofa MRPL45 K288 MRPL32 K132 S. scrofa MRPL12 K125 MRPL53 K14 S. scrofa * depending on isoform 3
4 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 2 (continued) 1 2 Organism ICT1 K198 MRPL18 K114 B. taurus MRPL53 K105 MRPL11 K13 B. taurus MRPL22 K48 MRPL24 K100 B. taurus MRPL12 K142 MRPL11 K13 B. taurus MRPL47 K164 MRPL28 K221 B. taurus MRPL11 K13 MRPL12 K138 B. taurus ICT1 K66 MRPL15 K239 B. taurus MRPL9 K81 MRPL28 K55 B. taurus MRPL39 K123 MRPL45 K302 B. taurus MRPL37 K40 MRPL28 K150 B. taurus MRPL9 K81 MRPL28 K51 B. taurus MRPL3 K279 MRPL19 K97 B. taurus MRPL10 K132 MRPL53 K25 B. taurus MRPL45 K302 MRPL39 K65 B. taurus MRPL16 K188 MRPL1 K71 B. taurus MRPL53 K41 MRPL11 K13 B. taurus MRPL22 K48 MRPL24 K100 B. taurus MRPL42 K99 MRPL39 K201 B. taurus MRPL37 K40 MRPL2 K71 B. taurus MRPL10 K57 MRPL57 K32 B. taurus MRPL45 K177 MRPL22 K48 B. taurus MRPL53 K105 MRPL11 K13 B. taurus MRPL12 K138 MRPL10 K162 B. taurus MRPL53 K41 MRPL36 K90 B. taurus MRPL12 K142 MRPL10 K162 B. taurus MRPL11 K106 MRPL16 K116 B. taurus MRPL11 K106 MRPL54 K106 B. taurus ICT1 K47 CRIF1 K113 B. taurus ICT1 K94 MRPL49 K158 B. taurus MRPL53 K105 MRPL12 K138 B. taurus MRPL32 K143 MRPL45 K302 B. taurus MRPL9 K158 MRPL1 K71 B. taurus MRPL47 K74 MRPL41 K69 B. taurus MRPL9 K207 CRIF1 K113 B. taurus 4
5 RESEARCH Supplementary Table 3 Crosslink data supporting our assignment of the localization of mitoribosomal-specific proteins. M r, theoretical molecular mass; m/z, experimentally determined mass-to-charge ratio; Id score, identification score calculated by xquest. Peptides FAFLLFKQDK-NALKSK-a7-b4 TFHTFDFYKK-NALKSK-a9-b4 ISSAIKTSR-KHLYSTR-a6-b1 EHFGEESGPKDR- LQEEEGKQK-a10-b7 TFHTFDFYKK-LQEEEGKQKa9-b7 EHFGEESGPKDRVDIGLPPPK- LQEEEGKQK-a10-b7 EHFGEESGPKDR- QQEKLQEEEGK-a10-b4 TYREHFGEESGPKDR- LQEEEGKQK-a13-b7 TFHTFDFYKK-QKNALK-a9-b2 SSGPGGQNVNKVNSK- GYFDQQLKAWLLEK-a11-b8 DMIAEASQPAKEPSR- VKMKPKPWSKR-a11-b6 KYGYVLPDITKDELFK- LPDDAKQANDIPVDR-a11-b6 INSAIKTSR-KHLYSTK-a6-b1 VPGDAKQGNDDIPVDR- LELAKK-a6-b5 SSGPGGQNVNKVNSK- GYFDQQLKAWLLEK-a11-b8 Central Protuberance * K274 K111 K85 K105 K305 K105 K85 K105 K85 K98 K85 K105 K305 K107 K305 K111 Placement of ICT1 Sus scrofa ICT1 MRPL18 K197 K114 ICT1 MRPL49 K93 K158 ICT1 MRPL19 K165 K227 MRPL15 K249 ICT1 K66 Bos taurus ICT1 MRPL18 K198 K114 ICT1 MRPL15 K66 K239 ICT1 MRPL49 K94 K158 M r (Da) m/z Id Score
6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 3 (continued) Peptides VQEVLKHAR-FQKELER-a6-b3 YGMSRPGSLSDKR- KAGLVIPHER-a12-b1 KAGLVIPHER-YVGKTVDYRa1-b4 KGASVDVDVR-SEKEFVEHARa1-b3 SCEIKFLTFK-EAHKPQLA-a5- b4 EAHKPQLA-IEVKHVGK-a4-b4 LHTLVTENCFPDMVWDIKYK- NKIVYPPQLPGEPR-a18-b2 Polypeptide Exit Site Sus scrofa MRPL20 MRPL44 K26 K43 MRPL24 MRPL45 K32 K62 MRPL45 MRPL24 K62 K100 MRPL23 MRPL45 K115 K54 Bos taurus MRPL39 MRPL45 K123 K302 MRPL45 MRPL39 K302 K65 MRPL45 K177 MRPL22 K48 M r (Da) m/z Id Score * MRPL38 K85 is not included in the MRPL38 homology model and its crosslinks are therefore not mapped in Figure 4. The crosslink between ICT1 and MRPL19 observed in S. scrofa but not B. taurus exhibits a very high ds score (ds = 0.9; ratio between the best and second best scoring peptide match) and therefore likely represents a false positive. see also Extended Data Fig
7 RESEARCH Supplementary Information References 1 Nakao, A., Yoshihama, M. & Kenmochi, N. RPG: The Ribosomal Protein Gene Database. Nucleic Acids Res. 32, (2004). 2 Kelley, L. A. & Sternberg, M. J. E. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4, (2009). 7
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