Cryo-EM and Computational Biology of Macromolecular Complexes

Abstract: The ribosome is a large, ancient multicomponent macromolecular complex which is highly amenable to study by cryogenic electron microscopy (cryo-EM) and computation biology methods. This thesis delves into the structure of both prokaryotic and eukaryotic ribosomes in the context of determining a solution to emerging antimicrobial resistance. We show that thermorubin (THB) binds to the E. coli ribosome at intersubunit bridge B2a, flipping out 23S rRNA residue C1914 which interferes with A-site substrates. The position and rearrangements caused by THB also accounts for the biochemical results showing a decrease in elongation, termination and recycling phases of translation. Also using cryo-EM we looked at the Giardia intestinalis ribosome, determining six high-resolution structures representing translocation intermediates. Giardia is a protozoan parasite causing diarrhoea in humans, with metronidazole strains emerging. As the ribosome is often a target for antimicrobial drugs, work on the structure and function of the ribosome is of utmost important in determining an alternative therapeutic approach to the treatment of giardiasis. We also show naturally bound tRNAs and eEF2 on the Giardia ribosome, exhibiting eukaryote-specific subunit rolling and eEF2 with GDP in a uniquely positioned Pi primed for release, adding to the mechanism of translocation in protists as well as illustrating the evolution of both the structure and function of translation machinery. Finally, the molecular basis of thermostability in translational GTPases is explored using molecular dynamics of mesophilic and thermophilic elongation factor EF-Tu. Through ancestral sequence reconstruction two key interactions: in the GTPase domain; and an interdomain interaction were shown to be important in the overall structural stability of EF-Tu in high temperature environments. These studies together highlight the strength of utilising both structural and computational techniques to explore the translation apparatus.