Strategies for High-Resolution Investigation of Bacterial Ribosome Function by Cryo-Electron Microscopy

This dissertation comprises projects focused broadly on cryo-electron microscopy (cryo-EM) methods and their application to high-resolution structural biology of the ribosome. The ribosome is the cell’s machinery for creating proteins in a series of steps referred to as translation. During translation, the large and small subunits of the ribosome participate in a cyclical process of association and disassociation, complexing with messenger RNAs, transfer RNAs, and many other translation factors along the way. From the intricacy of this process and of the ribosome itself comes a high degree of efficiency and fidelity in the synthesis of polypeptides. Structure is at the core of our current (and evolving) understanding of how the ribosome accomplishes this feat. Structures of the ribosome specifically are also rich with information implicating the fundamentals of RNA structure and folding, ribosomal assembly, medicine, and evolutionary relationships.Much of what has historically made an ideal specimen for single-particle cryo-EM is embodied by the ribosome: it is large, measuring approximately 25 nm in diameter and weighing approximately 2.6 MDa in bacteria, and it displays conformational flexibility that is difficult to capture and resolve by other methods. The recent widespread use of direct electron detectors has pushed the limits of what is achievable by single-particle cryo-EM, whose development over the past several decades has been punctuated by important advances involving the ribosome. Herein are described multiple stories from both sides of the coin – cryo-EM and ribosomal biology – including the most well-resolved ribosome structure to date, whose global resolution broke the 2 Å barrier for the first time for a large ribosomal subunit. Concluding thoughts consider future directions for cryo-EM methods development, informed by a specialized focus on the ribosome during a period of rapid growth in the field