Fig. 4 |. Cryo-EM structure of wild-type and ac⁴C-deficient T. kodakarensis ribosomes.

a, Ac⁴C distribution as observed by a cryo-EM image of wild-type T. kodakarensis grown at 85 °C. Modified residues are highlighted in orange, rRNA in grey and r-proteins are contoured in black. b, c, Ac⁴Cs participate in Watson–Crick pairing with guanine residues. b, An example of ac⁴C density shown in mesh. Residues correspond to ac⁴C2159 and G2725 of LSU. Acetate is highlighted yellow and is indicated by an arrow. c, The same position in the ΔTkNAT10 strain indicates that, in the mutant, the acetyl moiety is replaced by a structured solvent molecule. d, Ac⁴C in T. kodakarensis ribosomes derived from archaea grown at different temperatures, identified by ac⁴C-seq and LC–MS. e, ‘Core’ ac⁴Cs (shown in red) present at high stoichiometries across temperatures are enriched in the intersubunit interface and are in proximity to eL41 and to the ribosomal substrates. The functional ribosome regions indicated are the decoding centre (DC), the peptidyl-transferase centre (PTC) and the protein exit tunnel (ET). tRNA and mRNA are highlighted yellow, eL41 is shown in purple. The tRNA and mRNA coordinates are from PDB structure 4V5D. f, Misincorporation at core and auxiliary sites from T. kodakarensis and their conserved counterparts in P. furiosus and T. sp. AM4, grown at optimal growth temperatures (85 °C for T. kodakarensis and T. sp. AM4 and 95 °C for P. furiosus). n = 4 and 1 independent biological samples for T. kodakarensis and other archaea, respectively. Box plot visualization parameters are as in Fig. 1h. g, A representative example of the electrostatic interaction between ac⁴C and ribosomal proteins is shown between O(7) of ac⁴C1459 at h45 of small-subunit (SSU) and R15 of eL41 (top). The same position in the ΔTkNAT10 strain (bottom) implicates a solvent molecule that serves to mediate the same interaction network in the absence of an acetyl group. h, Thermal melting curves of synthetic RNA hairpin containing C (black) or ac⁴C (red) obtained by differential scanning calorimetry (DSC). Cp, heat capacity; T_m, melting temperature. Data are mean ± s.d. of n = 3 independent experiments.