BPS2026 – In situ single-molecule structural biology of ribosomes using 2D-template matching

Translation of mRNA by ribosomes is the final step in the expression of the genetic code and is therefore subject to exquisite spatial and temporal control. Hundreds of proteins bind and have the potential to regulate the ribosome, yet besides a few well-characterized translation factors; their specific functions remain largely unknown. Current approaches to characterize the function of ribosome-associated proteins lack spatial context, obscure structural detail, and/or average away single-molecule information, making it difficult to characterize rare events. Determining structural information of individual ribosomes is key to understanding translational regulation, which is thought to be mediated by specific and rare ribosomal states. Recent advances in cryogenic electron microscopy (cryo-EM) and two-dimensional template matching (2DTM) have now made it possible to study individual ribosomes directly in their native context. I will describe how we have leveraged 2DTM to detect ribosome-associated proteins on individual ribosomes and map the distribution of translation states with single molecule precision, all inside the cell. This work represents one of the first steps toward in situ single-molecule structural biology, opening the door to new discoveries in translational regulation and other processes involving large molecular machines.