Seminar

Revealing the Mechanisms of Biosynthetic Machines using High-Resolution Cryo-EM

Sean Connell, PhD

The term “Resolution Revolution”, coined by Werner Kuhlbrandt in 2014 (Science 343:1443), describes the profound impact new technologies, including direct electron detectors, improved electron microscopes, and novel computational methods, have had on the cryo-EM field as evidenced by the marked increase in the resolution and number of cryo-EM derived structures. This has sparked a democratization movement (Nature Methods 13:607) to make equipment and computational methodologies more accessible and encourage wide-spread adoption of the technique. Here, I will present how we have harnessed these advances in high-resolution cryo-EM to understand the molecular mechanisms inherent to the process of late 30S ribosomal assembly (López-Alonso, Nucleic Acids Research 45:2179) and the early steps in the initiation process of protein synthesis (López-Alonso, Nucleic Acids Research under revision). Specifically, we will show how cryo-EM can be used to understand the inhibitory mechanism of emerging antibiotics; for example, revealing that GE81112 stabilizes the initiating ribosome in a conformation constant with the tRNA and initiation factors being bound in an unlocked state, which prevents progression of the initiation process. With respect to 30S assembly, we have used high-resolution cryo-EM to reveal how RsgA couples the activation of its GTPase pocket to the assembly state of the 30S subunit such that it acts a checkpoint during the late stages of ribosomal assembly. These results are examples of how the increasing power of cryo-EM can be used to understand the chemical basis for the interactions and reactions that drive molecular machines like the ribosome.