The 2.9 Å cryo-EM map of the E. coli ribosome–EF-Tu complex has been deposited in the Electron Microscopy Data Bank with accession code EMD-2847, the coordinates of the atomic model have been deposited in the Protein Data Bank under accession code 5AFI.

• Extended Data Figure 1: Aberration-corrected cryo-EM.
  

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  a, Exemplary Zemlin tableau (left) and phase diagram (right) as obtained for the present data set with the CEOS software by correcting electron optical aberrations using the C_s corrector. The resulting phase errors were less than 45° at ≤2.1 Å (that is, at scattering angles of 12 to 15 mrad) over up to 36 h of image acquisition. The main limiting aberration is axial coma (B2) and the next limiting aberration would be threefold astigmatism (A3). b, Local correction for the contrast transfer function. From micrographs (left) areas with individual ribosome particles (yellow frames) were extracted and local power spectra were computed for each of these areas by fast Fourier transform algorithms (FFT). Local power spectra were subjected to principal component analysis (PCA) and classification to average power spectra with similar contrast transfer function parameters that were obtained from different micrographs. Class averages of power spectra reveal an improved signal-to-noise ratio in Thon rings which are clearly visible up to 2.4 Å (right). c, Global power spectrum from a single micrograph showing Thon rings up to 3.5 Å.

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• Extended Data Figure 2: Hierarchical sorting of ribosome particle images.
  

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  Ribosome particles were sorted in three steps according to: (1) global ribosome conformation (C1), that is, 30S body rotation⁷; and (2) ligand occupancy (C2)³⁵ and particle quality (C3)³⁷(Methods). The asterisk denotes particles assigned to the largest 30S body rotations ≤−10° and ≥10° which contain particles with extreme 30S rotation angles, but also low-quality particle images.

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• Extended Data Figure 3: Resolution curves and model validation of the E. coli 70S ribosome–EF-Tu cryo-EM structure.
  

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  a, Fourier-shell correlation (FSC) curve (black) for the 70S ribosome cryo-EM reconstruction computed between the masked independent half-maps (half1 and half2) that were obtained by so-called ‘gold-standard’ refinement in RELION³⁷. The resolution of the cryo-EM reconstruction is ~2.9 Å according to the 0.143 criterion⁶³ (black dashed line). b, FSC curves computed between cryo-EM maps and model maps generated from refined atomic coordinates. The vertical black dashed line indicates the maximum resolution at which the full atomic models were refined. Black, the FSC curve between the final cryo-EM map (map) and the final model (model); blue, the FSC curve between half map 1 (half1) and the model obtained by refinement only against half map 1 (model1); red, the FSC curve between half map 1 and the model obtained by refinement only against half map 2 (model2). c, FSC curves (FSC_work) between reflections from solvent-flattened cryo-EM map and model as obtained by pseudo-crystallographic refinement of the complete ribosome model (mask1) and three sub-models using different masks corresponding to local variations in resolution (mask2–4; Methods) as shown in h. Coloured numbers indicate the highest resolution used in refinement with the respective mask as indicated by the colour code. For all refinements, the FSC is above the 0.5 threshold (black dashed line) in the highest-resolution shell. Differences to b result largely from solvent-flattening before Fourier transformation for refinement (Methods). d–g, CC_work and R_work as obtained by refinement using the respective mask (see labels). For a reliable resolution estimate CC_work (ref. 8) is expected to be >0.2 and R_work <0.51 in the highest-resolution shell. h, Isosurface representations of the mask used for local refinements; ‘%’ indicates the fraction of atoms of the complete model entailed in the refinement with the respective mask.

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• Extended Data Figure 4: Modifications in rRNA. Comparison between cryo-EM and X-ray crystallography.
  

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  a, Experimental densities. In each row density maps for the same type of rRNA modification are shown (from left to right): for the present cryo-EM map and for the current best resolved bacterial and archaeal ribosome maps determined by X-ray crystallography, that is, the bacterial 70S ribosome from E. coli (Eco70S) at 2.8 Å resolution⁵ (PDB IDs: 4TPA and 4TPB); the bacterial 70S ribosome from T. thermophilus (Tth70S) at 2.4 Å resolution⁹ (PDB IDs: 4RB5 and 4RB6); and the archaeal 50S subunit from Haloarcula marismortui (Hma70S) at 2.2 Å resolution¹⁰ (PDB ID: 1VQ0). E. coli numbering is used for bacterial ribosome structures. Locations of rRNA modifications as determined by biochemical data are marked by yellow circles, modifications not observed in the density maps are denoted by red arrows and the black arrow designates the non-planarity of dihydrouridine observed in the cryo-EM map. b, Model-based densities for m⁶₂A1518 and m⁶₂A1519 showing slight differences due to scattering properties. Densities were computed in CCTBX⁶⁴ at 2.65 Å resolution from our final model with atomic-displacement factors kept unchanged using electron (e⁻ scattering, purple) and X-ray scattering factors (X-ray scattering, blue), respectively. Map thresholds were normalized to show similar density levels for the electron-rich phosphate groups. Accordingly, the absence of densities for modifications in crystallographic maps also at higher resolutions may result from differences in electron and X-ray scattering and in data quality which is affected, for example, by local and global disorder.

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• Extended Data Figure 5: rRNA modifications in the E. coli 70S ribosome.
  

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  a, Stabilizing effects of 16S rRNA methyl groups in the P site of the decoding centre. Numbers in the overview (top left) mark the positions of close-ups (1–3), which show the interactions of the rRNA methyl groups with distances colour-encoded by dashed lines. Close-up 1: Stacking network of m²G966 and m⁵C967 stabilizing binding of initiator tRNA. Close-ups 2, 3: rRNA modifications impacting mRNA binding. The universally conserved bulky dimethylamine groups of m⁶₂A1518 and m⁶₂A1519 stabilize their direct environment by steric encumbrance explaining their requirement for correct packing of 16S rRNA helices 24a, 44 and 45⁶⁵. In particular, the dimethylamine of m⁶₂A1519 is involved in medium and long-range repulsive interactions with the backbone of m³U1498 and the 2' O of C1520, while its conformation is mostly determined by the dimethylamine moiety of the adjacent m⁶₂A1518 which, in turn, is fixed by short repulsive interactions with O6 of G1517 and O4 of U793. The additional methyl groups of m⁶₂A1519 interact with A792, which provides part of the binding site for the antibiotic kasugamycin¹⁵, accounting for the resistance against kasugamycin upon demethylation of m⁶₂A1519⁶⁶. Furthermore, m⁶₂A1518 and m⁶₂A1519 impact initiation⁶⁷ possibly via m³U1498 whose backbone interacts with m⁶₂A1519, while its modified base contacts the mRNA backbone. The methyl groups of m³U1498 and m⁴Cm1402 form part of the binding site for the initiation codon and modulate translation initiation^{13, 14} by steric encumbrance and/or by preventing direct hydrogen bonds with the mRNA backbone. b, Constitutive rRNA modifications in the E. coli 70S ribosome (list adapted from ref. 11 and references therein). c, rRNA modifications in the A site of the decoding centre and helix 69 of 23S rRNA (H69). The binding site of aminoglycosides in helix 44 of 16S rRNA (h44)—including N4 of m⁵C1407—is indicated for neomycin B (magenta, superposition from PDB ID: 2ET4)⁶⁸. The three pseudouridines stabilizing H69⁶⁹ by enhancing base stacking⁷⁰ are depicted in blue. The methyl group (yellow) on m³Ψ1917 in H69 prevents potential base-pairing with A1913, a residue important for uniform tRNA selection⁷¹. Note the flipped out conformation of A1913 facilitating interaction with the 2′ OH of m²s⁶iA37 of the distorted Phe–tRNA^Phe (purple), which, in turn, stacks onto A36 of the tRNA anticodon. d, Methyl group on m²G1835 of 23S rRNA enhancing subunit association. The four helices of 23S rRNA that intersect around residue m²G1835 and form intersubunit bridges B2b and B2c with helices 24 (h24) and 45 (h45) of 16S rRNA (dark grey) are denoted in different colours: helix 67 (H67), light blue; helix 68 (H68), blue; helix 69 (H69), teal; helix 70 (H70), purple. Inset, contacts of the methyl group on m²G1835 with adjacent residues which, in turn, interact with 16S rRNA. e, Cluster of 23S rRNA modifications in the peptide exit tunnel. The modified rRNA residues, the functionally important nearby tip of protein L22 (teal), P-site fMet–tRNA^fMet (green) and a model of the nascent peptide chain (pink, superposition from PDB ID: 2WWL)⁷² are indicated.

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• Extended Data Figure 6: Visualization of structural dynamics of the ribosome by different approaches.
  

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  a–d, In each panel, the ribosome is shown from the factor binding site on the left and in cut-away view on the right; h denotes the head and b the body of the 30S ribosomal subunit. a, Present cryo-EM map coloured according to local resolution as determined by Resmap². b, Present cryo-EM map coloured according to the B factors obtained from the pseudo-crystallographic atomic model refinement (Methods). c, Model map of the 2.95 Å crystal structure of the E. coli 70S ribosome⁷³ (PDB IDs: 4KJ1 and 4KJ2) coloured according to respective B factors. The black arrow denotes the stabilization of the 30S head region by crystal contacts, whereas the 30S body (white arrow) is less constrained by crystal contacts and shows higher B factors, indicating larger flexibility for this region. d, Snapshot from molecular dynamics trajectory of the E. coli 70S ribosome coloured according to root mean squared fluctuations (RMSFs) obtained from the full 2 µs explicit solvent molecular dynamics simulation (Methods). Note the large fluctuations of the 30S head and body (white arrows) of the ribosome in solution not constrained by crystal contacts.

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• Extended Data Figure 7: Structure of E. coli EF-Tu–Phe–tRNA^Phe bound to the ribosome.
  

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  a, Detailed comparison of the distorted A/T-site–tRNA interactions between the E. coli and T. thermophilus ribosome–EF-Tu–kirromycin complexes^{6, 26} and the free E. coli tRNA^Phe51. We found significant differences in tRNA conformation and interactions implicated in the GTPase activation mechanism²⁶ that correlate with ribosome binding and differences in organism and tRNA species. Here and below residue numbers refer to E. coli. b, Overview of the E. coli ribosome–EF-Tu structure. The residues interacting with the EF-Tu ternary complex (depicted in stick representation) generally agree with those seen in the T. thermophilus structures^{6, 26}. rRNA helices are denoted as: h44, helix 44 of 16S rRNA; H69, helix 69; and SRL, sarcin–ricin loop of 23S rRNA. The dashed boxes indicate the parts of the structure magnified in c and d. c, Structural differences in an important ribosome–EF-Tu interaction. In the E. coli structure (Eco, left panel) residues A55 and A368 of 16S rRNA assume different conformations (cyan arrows) and interact differently with the ribosome and EF-Tu–tRNA complex than in the T. thermophilus structure (Tth, right panel, PDB ID: 2WRN)²⁶. Furthermore, the differences in EF-Tu sequence result in slightly different ribosome–EF-Tu interactions, for example, the ε-amino group of lysine 282 in E. coli EF-Tu is within hydrogen-bonding distance of G382 in 16S rRNA, but not the serine at this position in T. thermophilus EF-Tu. Inset on left panel, overlay of the crucial β-turn^{26, 74} in EF-Tu domain 2 in the free (PDB ID: 1OB2, R. C. Nielsen et al., unpublished data) and ribosome-bound state from E. coli and T. thermophilus. d, Dynamics of the catalytic histidine 84 (ref. 75) of EF-Tu. A split density for the side chain of histidine 84 (data not shown) indicates the presence of two rotamers (rot1 and rot2, panel 1) in the present E. coli complex. In d, the T. thermophilus ribosome-bound EF-Tu structures^{6, 26, 76} (dark grey, complex as indicated) are shown with the corresponding rotamer of the present structure (red). Residues valine 20 and isoleucine 60 of the hydrophobic gate⁷⁶ are denoted; isoleucine 60 is not resolved in the kirromycin-stalled ribosome–EF-Tu complexes.

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• Extended Data Figure 8: Cryo-EM densities for mobile proteins L9 and L31 and the arrangement of L9 in polysomes.
  

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  Densities in a and b were obtained by semi-automatic segmentation using the ‘segger’ tool in UCSF CHIMERA^{41, 77} and normalized and low-pass filtered according to local resolution estimates. a, Cryo-EM map and models of protein L9 (~4 Å local resolution, rendered at 1σ). b, Cryo-EM maps and models of protein L31 in the ground-state of the ribosome (left, ~4.3 Å local resolution) and in the rotated state (right, ~6 Å local resolution); maps were rendered at 1.5σ. c, Model of protein L9 in the context of polysomes. Overviews show the arrangement of neighbouring ribosomes (i-1 and i) in the major t-t form of E. coli polysomes as obtained by fitting the present 70S ribosome structure into the cryo-electron tomography reconstruction²⁷ in UCSF CHIMERA⁴¹. Left close-up, the conformer of L9 as seen in the present cryo-EM map (L9 cryo-EM, blue) is located close to protein S4 of the neighbouring 30S subunit (30S i) according to the polysome model. The purple arrow indicates the rearrangement of L9 from the cryo-EM conformation to that seen in crystals. The black arrow denotes the location of the mRNA entry channel in the 30S subunit i. Right close-up, the conformer of L9 as seen in the context of ribosome crystals (L9 X-ray, pink) reaches into the ribosomal A-site of the neighbouring 30S subunit and would be compatible with the simultaneous binding of elongation factors in the polysome model. In crystals, protein L9 precludes the binding of elongation factors due to the tighter packing of ribosomes⁷⁸. The model of L9 was obtained by superposition of the E. coli 70S ribosome X-ray structure⁵ (PDB IDs: 4TP8 and 4TP9) onto ribosome i-1 in the polysome model using UCSF CHIMERA⁴¹.

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• Extended Data Table 1: Data collection and model refinement
  

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  *For model refinement, the cryo-EM map was cropped from 420 × 420 × 420 pixels to 400 × 400 × 400 pixels.

  †Refinement target: MLHL maximum likelihood with experimental phase probability distribution.

  ‡Highest resolution shell is shown in parenthesis.

  § , where F_EM are structure factors calculated based on the solvent-flattened cryo-EM map and F_model are structure factors calculated from the refined model. The structure factors belonged to working set which was used for reciprocal space refinement.

  ∥CC_work = Pearson correlation coefficient calculated between F_EM and F_model.

  ¶FSC_work = is the averaged over all shells Fourier Shell Correlation (FSC_overall) calculated between F_EM and F_model belonging to the working set. The value for the highest resolution shell is shown in parenthesis and was calculated using:

  

  #Calculated with RESOLVE⁵⁴.

  ⋆Residue averaged real space local correlation coefficient (RSCC) in the region of the model to the cryo-EM map calculated with RESOLVE.

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• Extended Data Table 2: Comparison of densities as observed in cryo-EM and X-ray crystallography by local cross-correlation*
  

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  *Real space correlation coefficients (RSCCs) were calculated with RESOLVE⁵⁴ to compare the local model fit to the normalized experimental map.

  †‘acceptable density’: no atom lies in density <1 s.d. below 1/2 mean for that atom type and no group is <1/2 mean for that group.

  ‡‘residues with some weak density’: all residues which do not fulfill the requirement for acceptable density.

  §‘out of density’: density level <2 standard deviations below 1/2 mean density for the particular residue.

  ∥‘very weak density’: density level <1 s.d. below 1/2 mean density for the particular residue.

  ¶‘weak density’: density level <1/2 mean density for the particular residue.

  Overall, the definition of protein and RNA main and side chain densities is similar between the present cryo-EM data and 70S ribosome crystal structures at similar resolutions, suggesting also a similar extent of radiation damage. The RSCC values obtained for specific amino acid side chain groups indicate different sensitivity to radiation damage for specific side chains, with negatively charged side chains being the most sensitive ones both in cryo-EM and X-ray crystallography^{60, 61, 62}. The elevated RSCCs calculated for the 2.95 Å E. coli 70S–EF-G crystal structure show the smallest fluctuations between different side chain groups and, thus, the lowest level of radiation damage, most probably owing to multi-crystal merging of 20 partial data sets. In contrast, experimental data for the 2.8 Å E. coli 70S–quinupristin and the 2.8 Å T. thermophilus 70S–tRNA crystal structures were collected from two and one crystal(s), respectively.

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