Abstract

Ribosomes are the supramolecular complexes responsible for protein synthesis. The large 50S ribosomal subunit catalyzes the peptidyl transferase reaction and peptide bond formation between amino acids. The 50S is targeted by many known clinically effective antibiotics. Available structures, obtained at cryogenic temperatures (CT), are used for drug discovery despite that active or important target sites may display a structural configuration that is CT-induced. The introduction of ultrafast and ultrabright X-ray free electron laser (XFEL) pulses has enabled the structural observation of biological macro- and supramolecules at previously unattainable, near-physiological temperatures. In this study, we use ultrafast and ultrabright XFEL pulses to solve the apo form of 50S ribosomal subunit isolated from the extremely thermophilic bacterium Thermus thermophilus at ambient temperature (AT). The dimeric structure of the 50S subunit presented in this work is among the largest ([~]3 megadalton) structures determined using an XFEL source to date. This study demonstrates the ability to obtain new information about ribosome structural dynamics at AT through serial femtosecond X-ray crystallography (SFX). This allowed us to capture previously unobserved dynamics of ribosomal protein uL23 and coordination by hexahydrated magnesium cations at a hitherto unseen resolution at near-physiological temperature. Also, residue A2602, at the core of the peptidyl transferase center (PTC), shows a rather different orientation of the sugar moiety if compared to CT structures. In addition, our structure highlights the importance of flexible residues at both the PTC and in the binding sites for antibiotics erythromycin and chloramphenicol. The method implemented here may also serve as a starting point for future structural research involving the 50S subunit complexes by employing time-resolved mix-inject and probe kineto-crystallography experiments at XFELs. Unveiling ligand-dependent 50S dynamics at physiological temperatures shall guide further development of next-generation antibiotics that target the translation machinery.