Generation and maintenance of mammalian cell lines

The Flp-In-TRex human embryonic kidney 293T (HEK293T) cell line (Invitrogen) was cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) tetracycline-free FBS, 2 mM Glutamax (Gibco), 1× penicillin–streptomycin (Gibco), 50 µg ml−1 uridine, 10 µg ml−1 Zeocin (Invitrogen) and 100 µg ml−1 blasticidin (Gibco) at 37 °C under 5% CO2 atmosphere. Cell lines have been routinely tested for mycoplasma contamination.

For SILAC-based quantitative proteomic analysis, cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) for Stable Isotope Labeling by Amino acids in Cell culture (SILAC) supplemented with ‘heavy’ (15N-labelled and 13C-labelled) or ‘light’ Arg, Lys and Pro and 10% dialysed FCS (Thermo Scientific HyClone).

The Trmt2b-knockout cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), GlutaMAX (Gibco) and penicillin–streptomycin (Gibco) at 37 °C, 5% CO2. Large cell culture volumes were grown in Thomson OptimumGrowth 1.6-l and 5-l flasks in a shaker incubator at 110 rpm. For the cryo-EM sample, approximately 10 bln cells of the Trmt2b-knockout cell line were used.

Trmt2b gene inactivation

For the mitoribosome preparation, we used an NS0 mouse cell line with an inactivated Trmt2b gene that has been characterized in ref. 16. In brief, guide RNA (gRNA) for Cas9 targeting the first coding exon of Trmt2b was inserted into the pX458 vector (Addgene #48138). NS0 cells were transfected with the pX458 plasmid containing the gRNA sequence using a Lipofectamine 3000 reagent (Thermo Scientific). Twenty-four hours after transfection, GFP-positive cells were sorted using BD FACS Aria III in 96-well plates containing 0.2 ml of RPMI medium per well. Individual clones were analysed by PCR amplification of an approximately 250-bp Trmt2b fragment (5ʹ-TCAAGAGTCCTAAATGCACAACC-3ʹ and 5ʹ-CCAGGAGTCATCTCTACAATGC-3ʹ) and sequencing of the amplicon. For off-target analysis, we chose five off-targets with the highest score according to the Benchling CRISPR gRNA designing tool (https://benchling.com). Each off-target was analysed by PCR amplification of approximately 250 bp and sequencing.

MRM3, mS37 and GTPBP10 gene inactivation

To generate the MRM3-knockout and mS37-knockout cell lines, two pairs of gRNAs targeting exon 1 of MRM3 or mS37 (Supplementary Table 4) were designed and cloned into the pSpCas9(BB)-2A-Puro (pX459) V2.0 vector to generate out-of-frame deletions. Cells were transfected with the pX459 variants using Lipofectamine 3000 according to the manufacturer’s recommendations. Transfected cell populations were selected by puromycin treatment at a final concentration of 1.5 µg ml−1 for 48 h. Subsequent to this, cells were diluted to achieve single-cell-derived clones on 96-well plates. Resultant clones were screened by Sanger sequencing to assess knockout, and loss of MRM3 and mS37 in selected clones was confirmed by western blotting. The GTPBP10-knockout cell line was generated as previously described34.

Generation of the rescue cell lines

To generate knockout cell lines stably re-expressing the protein of interest (mS37, MRM3 and GTPBP10), the HEK293T cell line that permits doxycycline-inducible expression of the gene of interest in a dose-dependent manner was used. HEK293T cells were cultured at 37 °C under 5% CO2 in DMEM supplemented with 10% (v/v) tetracycline-free FBS, 1× penicillin–streptomycin (Gibco), 50 μg ml−1 uridine, 100 μg ml−1 zeocin (Invitrogen) and 10 μg ml−1 blasticidin S (Gibco). Knockout cells were seeded in a 10-cm dish, 1 day before transfection, to achieve 70–90% confluency. Co-transfection of the expression plasmid pcDN5/FRT/TO (with mS37, MRM3-Flag and GTPBP10-Flag) and the Flp-recombinase plasmid pOG44 was carried out using Lipofectamine 3000 (Invitrogen), according to the manufacturer’s instructions. Selective antibiotics, 100 μg ml−1 of hygromycin (Invitrogen) and 10 μg ml−1 blasticidin S were added 48 h post-transfection and culture media were replaced every 2–3 days. To induce expression of the protein of interest, cells were incubated with 50 ng ml−1 doxycycline for 48 h before analysis.

Mitoribosome preparation

Cells were harvested by centrifugation at 1,000g for 7 min. Cells were resuspended in MIB buffer (50 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) and allowed to swell by stirring on ice. SM4 buffer (0.28 M sucrose, 0.84 M mannitol, 50 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) was added to adjust the final concentrations of sucrose and mannitol to 70 mM and 0.21 M, respectively. Lysis of the cells was done in a nitrogen cavitation chamber at 500 psi for 20 min on ice. Lysate was cleared by centrifugation at 800g for 15 min. Supernatant was collected and the pellet was resuspended in half of the volume of the MIBSM buffer (70 mM sucrose, 0.21 M mannitol, 50 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) used at the previous step and homogenized in teflon Dounce homogenizer. Homogenate was centrifuged 800g for 15 min and supernatant was collected. The procedure with the pellet was repeated using half of the MIBSM buffer volume in the previous step. All supernatants were combined. Mitochondria were pelleted from cell lysates by centrifugation at 10,000g for 15 min at 4 °C, resuspended in MIBSM buffer and loaded on top of the step sucrose gradient (1.0 M and 1.5 M sucrose in 20 mM HEPES-KOH pH 7.5, and 1 mM EDTA) in SW40 tubes (Beckman Coulter) and centrifuged in a SW40 Ti rotor at 28,000 rpm (139,000g) for 1 h at 4 °C. Mitochondria were collected from tubes, resuspended in equal volume of 20 mM HEPES-KOH pH 7.5, and centrifuged at 10,000g for 10 min at 4 °C. Pellets of mitochondria were snap-frozen in liquid nitrogen and stored at –80 °C.

Purified mitochondria were defrosted and lysed in two volumes of lysis buffer (25 mM HEPES-KOH pH 7.5, 100 mM KCl, 20 mM Mg(OAc)2, 2% Triton X-100, 2 mM DTT, 1X cOmplete EDTA-free protease inhibitor cocktail (Roche) and 40 U μl−1 RNase inhibitor (Invitrogen) for 10 min on ice. Lysates were cleared by centrifugation at 15,871g for 10 min at 4 °C, then each 1 ml was loaded on top of a 0.4-ml sucrose cushion (0.6 M sucrose, 25 mM HEPES-KOH pH 7.5, 50 mM KCl, 10 mM Mg(OAc)2, 0.5% Triton X-100 and 2 mM DTT) and centrifuged in a TLA 120.2 rotor (Beckman Coulter) at 100,000 rpm (436,000g) for 1 h at 4 °C. The pellet was resuspended in resuspension buffer (25 mM HEPES-KOH pH 7.5, 50 mM KCl, 10 mM Mg(OAc)2, 0.05% n-dodecyl β-d-maltoside (β-DDM) and 2 mM DTT), loaded on top of 10 ml of a 15–30% sucrose gradient (25 mM HEPES-KOH pH 7.5, 50 mM KCl, 10 mM Mg(OAc)2, 0.05% β-DDM and 2 mM DTT) and centrifuged in a TLS-55 rotor (Beckman Coulter) at 39,000 rpm (130,000g) for 2 h 15 min. Mitoribosome gradients were fractionated into approximately 100-µl fractions and absorbance of each fraction was measured. Fractions corresponding to SSU and LSU peaks were gathered and the buffer was changed to resuspension buffer using centrifugal concentrator Vivaspin MWCO 30,000 PES (Sartoius). Obtained SSU and LSU solutions were used for grid preparation.

SDS–PAGE and western blot analysis

To assess the steady-state levels of the individual proteins, total cell extracts or purified mitochondria were analysed using the SDS–PAGE and western blotting. Cell pellets from the wild-type HEK293T cells and MRM3-knockout, mS37-knockout and GTPBP10-knockout lines were lysed (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100 and 1× PIC (Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts (approximately 30 μg) of total cell extracts or mitochondrial proteins (approximately 10 μg) were separated by SDS–PAGE and subsequently wet transferred to the polyvinylidene difluoride (PVDF) membranes for western blotting. The membranes were blocked for 1 h with 5% non-fat milk (Semper) in PBS and further incubated overnight with specific primary antibody at 4 °C. The next day, the blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody and visualized using enhanced chemiluminescence (ECL; Bio-Rad). The primary and secondary antibodies are summarized in Supplementary Table 4.

[35S]-metabolic labelling of mitochondrial proteins

To label newly synthesized mitochondrial DNA-encoded proteins, cells were seeded into a six-well dish at 80–90% confluency. First, two washing steps of 5 min each in methionine/cysteine-free DMEM were performed. Subsequently, cells were incubated with fresh methionine/cysteine-free DMEM supplemented with Glutamax 100X (Gibco), sodium pyruvate 100X (Gibco), 10% dialysed FBS and 100 µg ml−1 emetine (Sigma-Aldrich) for 20 min at 37 °C. Labelling was performed with the addition of 166,7 µCi ml−1 of EasyTag EXPRESS [35S] protein labelling mix (methionine and cysteine) (Perkin Elmer) for 30 min at 37 °C. Following labelling, cells were washed with 1 ml of PBS three times and the final pellets were collected by centrifugation. Cells were lysed in 1× PBS-PIC with the addition of 50 units of benzonase (Life Technologies) with incubation on ice for 20 min, followed by the addition of SDS to 1% final concentration. and further incubation on ice for 30 min. After cell lysis, 30 µg total protein was separated on Bolt 12% Bis-Tris Plus (Invitrogen) SDS–PAGE gels. Gels were then incubated in Imperial Protein Stain (Thermo Fisher) for 1 h and with fixing solution (20% methanol, 7% acetic acid and 3% glycerol) for 1 h. Next, gels were vacuum-dried at 65 °C for 2 h. The resultant gel was exposed to storage phosphor screens and visualized with Typhoon FLA 7000 Phosphorimager.

Sucrose gradient centrifugation analysis

Isolation of mitochondria and sucrose gradient centrifugation was performed as previously described2. In brief, 1 mg of mitochondria was lysed in lysis buffer (10 mM Tris-HCl pH 7.5, 100 mM KCl or 50 mM KCl, 20 mM MgCl2, 1× PIC, 260 mM sucrose and 1% Triton X-100) freshly supplemented with 0.4 U µl−1 final concentration of RNase Block Ribonuclease Inhibitor (Agilent), loaded onto a linear sucrose gradient (10–30%, 11 ml total volume) in 1× gradient buffer (20 mM Tris-HCl pH 7.5, 50 mM KCl, 20 mM MgCl2 and 1× PIC) and centrifuged for 15 h at 79,000g at 4 °C (Beckman Coulter SW41-Ti rotor). A total of 25 fractions with a volume of 450 µl each were collected via pipetting from the top of the gradient, and 15 µl of each fraction was used for western blot analysis. For fractions 1 and 2, and for fractions 18 and 19, 7.5 µl of each fraction was combined and resolved together.

For SILAC-based proteomics, HEK293T-knockout or MRM3-kncokout cells were grown in ‘heavy’, containing 15N-labelled and 13C-labelled Arg and Lys, or ‘light’-labelled media for more than seven doublings. Cell lines were pooled together, and mitochondrial isolation and sucrose gradient analysis were performed as described above.

Mass spectrometry analysis

Peptides from SILAC sucrose gradient centrifugation experiments were prepared from fractions 1 and 2 joined, 3 and 4 joined, and 5 to 17 individually. Collected fractions were precipitated in 20× 100% ice-cold ethanol overnight at –20 °C. Pelleted proteins were resuspended in 6 M GuHCl/Tris pH 8.0 solution and sonicated for 5 min at maximum output (10-s on/off cycles). After a 5-min incubation at room temperature, samples underwent a second round of sonication and were later centrifuged at maximum speed for 10 min. DTT, at a final concentration of 5 mM, was added to the obtained supernatants and incubated for 30 min at 55 °C followed by incubation with 15 mM chloroacetamide for 15 min at room temperature in the dark. Before digestion, protein quantification was performed and trypsin (Pierce, trypsin protease MS-grade, Thermo Fisher Scientific) was added accordingly. Protein digestion was performed at 37 °C overnight, mildly shaking. After 12–14 h, trypsin was inactivated using 1.2% formic acid and samples were spinned down at 3,000g for 10 min at room temperature. Samples were desalted, using desalting columns (Thermo Fisher Scientific) previously equilibrated and washed respectively with 100% acetonitrile and 0.5% formic acid, and eluted (0.5% formic acid and 50% acetonitrile). Peptides were consequently dried using SpeedVac Vacuum Concentrator and resuspended in 0.5% formic acid for mass spectrometry.

For liquid chromatography with tandem mass spectrometry analysis, peptides were separated on a 25-cm, 75-μm internal diameter PicoFrit analytical column (New Objective) packed with 1.9-μm ReproSil-Pur 120 C18-AQ media (Dr. Maisch) using an EASY-nLC 1000 (Thermo Fisher Scientific). The column was maintained at 50 °C. Buffer A and buffer B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile. Peptides were separated on a segmented gradient from 6% to 31% buffer B for 57 min and from 31% to 44% buffer B for 5 min at 250 nl min−1. Eluting peptides were analysed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurements were carried out at 60,000 resolution in the 350–1,500 m/z range. The most intense precursors with charge state from 2 to 7 only were selected for higher-energy collisional dissociation (HCD) fragmentation using 27% normalized collision energy. The m/z values of the peptide fragments were measured at a resolution of 50,000 using an automatic gain control (AGC) target of 2 × 10−5 and 86-ms maximum injection time. The cycle time was set to 1 s. Upon fragmentation, precursors were put on a dynamic exclusion list for 45 s.

For protein identification and quantification, the raw data were analysed with MaxQuant version 1.6.1.0 (ref. 36) using the integrated Andromeda search engine37. Peptide fragmentation spectra were searched against the canonical sequences of the human reference proteome (proteome ID UP000005640, downloaded September 2018 from UniProt). Methionine oxidation and protein N-terminal acetylation were set as variable modifications; cysteine carbamidomethylation was set as fixed modification. The digestion parameters were set to ‘specific’ and ‘Trypsin/P’. The minimum number of peptides and razor peptides for protein identification was 1; the minimum number of unique peptides was 0. Protein identification was performed at a peptide spectrum matches and protein false discovery rate of 0.01. The ‘second peptide’ option was on. Successful identifications were transferred between the fractions using the ‘Match between runs’ option. Differential expression analysis was performed using limma, version 3.34.9 (ref. 38) in R, version 3.4.3.

Cryo-EM data collection and image processing

For cryo-EM analysis, 3 μl of approximately 100 nM mitoribosome was applied onto a glow-discharged (20 mA for 30 s) holey-carbon grid (Quantifoil R2/1 or R2/2, copper, mesh 300) coated with continuous carbon (of approximately 3-nm thickness) and incubated for 30 s in a controlled environment of 100% humidity at 4 °C. The grids were blotted for 3 s, followed by plunge-freezing in liquid ethane, using a Vitrobot MKIV (FEI/Thermo Fisher). The datasets were collected on a FEI Titan Krios (FEI/Thermo Fisher) transmission electron microscope operated at 300 keV with a slit width of 20 eV on a GIF quantum energy filter (Gatan). A K2 Summit detector (Gatan) was used at a pixel size of 0.81 Å or 0.83 Å (magnification of ×165,000) with a dose of 30–32 electrons per Å2 fractionated over 20 frames. A defocus range of 0.8–3.8 μm was used. Detailed parameters are listed in Supplementary Tables 1 and 2.

Beam-induced motion correction was performed for all datasets using RELION 3.0 (ref. 39). Movie stacks were motion corrected and dose weighted using MotioCor2 (ref. 40). Motion-corrected micrographs were used for contrast transfer function (CTF) estimation with GCTF41. Particles were picked by Gautomatch (https://www.mrc-lmb.cam.ac.uk/kzhang) with reference-free, followed by reference-aided particle picking procedures. The reference-based pickings of SSU and LSU particles were done separately, using their corresponding picking references. Reference-free 2D classification was carried out to sort useful particles from falsely picked objects, which were then subjected to 3D refinement, followed by a 3D classification with local-angular search. UCFS Chimera42 was used to visualize and interpret the maps. 3D classes corresponding to unaligned or low-quality particles were removed. Well-resolved classes were pooled and subjected to 3D refinement and CTF refinement (beam-tilt, per-particle defocus and per-micrograph astigmatism) by RELION 3.1 (ref. 43), followed by Bayesian polishing. Particles were separated into multi-optics groups based on acquisition areas and date of data collection. A second round of 3D refinement and CTF refinement (beam-tilt, trefoil and fourth-order aberrations, magnification anisotropy, per-particle defocus and per-micrograph astigmatism) were performed, followed by 3D refinement.

To classify the SSU states, non-align focus 3D classifications with particle-signal subtraction using the mask covering the factor binding were done with RELION 3.1 (Extended Data Figs. 2 and 3). The particles of each state were pooled, the subtracted signal was reverted and 3D refinement was done with the corresponding solvent mask. To improve the local resolution, the several local masks were prepared and used for local-masked 3D refinements (Extended Data Figs. 2 and 3 and Supplementary Tables 1 and 2). Nominal resolutions are based on gold standard, applying the 0.143 criterion on the Fourier shell correlation between reconstructed half-maps. Maps were subjected to B-factor sharpening and local-resolution filtering by RELION 3.1, superposed to the overall map and combined for the model refinement.

Model building, refinement and analysis

The starting models for SSU was Protein Data Bank (PDB) ID 6RW4 (ref. 2), whereas those of LSU were PDB IDs 6ZM5 (ref. 4) and 5OOM44. These SSU and LSU models were rigid body fitted into the maps, followed by manual revision. Initial models of RBFA, TFB1M and METTL15 were generated by SWISS-MODEL45 using PDB IDs 2E7G, 4GC9 and 1WG8, respectively, as templates. Ligands and specific extensions or insertions were built manually based on the density. Secondary-structure information prediction by PSIPRED46 was also considered for low-resolution regions. mtIF3 and mtIF2 models were modified from previous work2 (PDB IDs 6RW4 and 6RW5). fMet-tRNAMet was from PDB ID 6GAZ (ref. 3) with the addition of the modification of f5C, whereas the mRNA was manually built into the density. The CTD of bL12m was generated from PDB ID 1CTF. Coot 0.9 with Ramachandran and torsion restraints47 was used for manual fitting and revision of the model.

Water molecules were automatically picked by Coot, followed by manual revision. Geometrical restraints of modified residues and ligands were calculated by Grade Web Server (http://grade.globalphasing.org) or obtained from the library of CCP4 7.0 (ref. 48). Hydrogens were added to the molecules except for waters by REFMAC5 (ref. 49).

Final models were subjected to refinement of energy minimization and atomic displacement parameters (ADP) estimation by Phenix.real_space_refine v1.18 (ref. 50) with rotamer restraints without Ramachandran restrains, against the composed maps with B-factor sharpening and local-resolution filtering. Reference restrains were also applied for non-modified protein residues, using the input models from Coot as the reference. Metal-coordination restrains were generated by ReadySet in the PHENIX suite and used for the refinement with some modifications. Non-canonical covalent bond restrains between non-modified residues and modified residue or ligand were prepared manually and used. Refined models were validated with MolProbity51 and EMRinger52 in the PHENIX suite. Model refinement statistics are listed in Supplementary Tables 1 and 2. UCSF ChimeraX 0.91 (ref. 53) was used to make the figures.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.



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