The translating bacterial ribosome at 1.55 Å resolution generated by cryo-EM imaging services

Abstract

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.

Anionic G•U pairs in bacterial ribosomal rRNAs

“…Compared with the standard wobble pair, the anionic G•U pairs display the U displaced into the shallow minor groove with a distance between the sugar carbon C1' larger by about 1 Å (see Table 1). The high quality of the recent structures (Watson et al 2020;Fromm et al 2023), together with the residue conservations in sequence alignments and their systematic occurrences in previous ribosomal structures, exclude a systematic error in molecular fitting in the observed anionic G•U pairs. The quality of the fits is illustrated by the cryo-EM maps of two anionic G•U pairs (Figures 7A, 7B) and the anionic G664•G741 pair (Figure 7C) are displayed.…”

“…The distances to the Mg 2+ ion are between 1.9 Å and 2.1 Å, except the one to the O6(G1099). Drawings based on E. coli (PDB 8B0X, (Fromm et al 2023)). (PDB 8B0X, (Fromm et al 2023)).…”

“…Drawings based on E. coli (PDB 8B0X, (Fromm et al 2023)). (PDB 8B0X, (Fromm et al 2023)). Please note that in 8B0X that pair is numbered G2308•U2316 (see Table 1).…”

“…Summary of the anionic base pairs observed in structures of bacterial ribosomes. The references for the PDB codes are the following: 7K00 (Watson et al 2020); 8B0X (Fromm et al 2023), 5E81 (Rozov et al 2015), 4Y40 (Polikanov et al 2015), 5NJT (Beckert et al 2017), 5NGM (Matzov et al 2017), 6S0Z (Halfon et al 2019).…”

“…We wondered whether such alternative G high resolution to be confident of the hydrogen bond distances and the relative positions of the nucleotides in G•U pairs. For E. coli ribosomes, high-resolution structures are now available at 2.0 Å (Watson et al 2020) and 1.55 Å (Fromm et al 2023).…”