To visualize the interactions of MyxA with the ribosome, we determined a cryo-EM structure of MyxA in complex with the E. coli ribosome at 2.10 Å (for the large 50S subunit) to 2.54 Å (for the complete 70S ribosome) (Fig. ,2 and Table -1). Additional density was observed within the nascent polypeptide exit tunnel (NPET) that could be unambiguously assigned to MyxA (Fig. , Fig. and Fig. ). While the cryo-EM density allowed the majority of MyxA to be modelled (Fig. and Fig. ), the D-Ala13, D-ß-OH-Val14 and terminal AG moiety were less well-resolved and observed only at lower map thresholds (Fig. ). Additionally, we observed density that we attributed to seven water molecules (W1-W7) (Fig. and Fig. ). The density for MyxA was consistent with the stereochemistry of Val7 and Val10 having D-and L-conformations, as determined here (Fig. ), rather than L-and D-conformations, as suggested previously (Fig. , f) . The overall conformation of MyxA on the ribosome is oriented with the MBA moiety positioned at the PTC and the AG extending down the NPET towards ribosomal protein L22 (Fig. ). The central region of MyxA is highly compacted, which is likely driven by stacking interactions from the sidechains of D-ß-Tyr6 and D-Arg9 of MyxA with the nucleobases of A2062 and G2505, respectively, of the 23S rRNA (Fig. and Fig. ). The compacted conformation is further stabilized by intramolecular interactions between D-Arg9 and backbone carboxyls of L-Ala2 and D-Val5 of MyxA (Fig. ). In addition, the sidechain OH group of D-ß-Tyr6 can hydrogen bond with the N7 of A2503 (Fig. ). This latter interaction, together with a potential hydrogen bond from the backbone carboxyl of dh-Val1 to the N2 of G2061 (Fig. ), represent the only direct hydrogen bonds that can be formed between MyxA and the rRNA, whereas we predict at least nine additional hydrogen bonds between MyxA and rRNA that are mediated via the water molecules W1-W7 (Fig. ). Unsurprisingly, given the highly hydrophobic nature of most of the MyxA sidechains (Ala, Val and Ile), the majority of the water-mediated interactions involve the backbone carboxyls of MyxA, with the exceptions including the polar sidechains of D-ß-Tyr6 (with W3) (Fig. ) and D-Arg9 (with W5 and W6) (Fig. ). The high structural conservation of the PTC and NPET between M. tuberculosis and E. coli 70S ribosomes suggests that MyxA is likely to bind to M. tuberculosis ribosomes in a similar fashion to that observed here for E. coli . Consistently, the MyxA R resistance mutations identified within the rRNA of M. tuberculosis all map directly within the binding site of MyxA on the E. coli 50S subunit (Fig. ). Mutations at position A2503G (MtuA2741G), U2609C (MtuU2847C) and C2611G (MtuU2849G) are predicted to cause conformational changes in the rRNA nucleotides that abolish direct and water-mediated interactions with MyxA (Fig. and Fig. ). The binding site of MyxA in the NPET overlaps with nascent polypeptide chains (Fig. ), suggesting that MyxA is likely to bind to the ribosome and inhibit translation before the polypeptide is synthesized. Indeed, superimposition of A-and P-site tRNAs reveals that the MBA and dh-Val1 moieties of MyxA would sterically clash with the aminoacyl moiety of an A-site tRNA accommodated at the PTC (Fig. ). Additionally, a slight clash between the D-Val4 of MyxA and the methionine sidechain of an initiator fMet-tRNA located at the P-site is predicted, suggesting that MyxA is likely to interfere with the final placement of the P-site tRNA during translation initiation.