Ribosome biogenesis requires hundreds of proteins and RNA assembly factors to form the small subunit (SSU) and large subunit (LSU) of the ribosome. Snapshots of this complex process have been captured by single particle cryogenic electron microscopy (SPA cryo-EM). However, how these structures are spatially organized within the nucleolus is unknown. To answer this question, we used high-resolution structures of SSU and LSU biogenesis intermediates and located them within the yeast nucleolus with two-dimensional template matching (2DTM) on 2D cryoEM images. Briefly, in 2DTM, a single exposure 2D image of the cell section is acquired (avoiding tilt angles). A 3D template, generated from a structural model, is used to create millions of 2D projections, which are then matched to our complexes of interest with high angular and positional accuracy using a fine-grained angular search. This process allowed us to map each intermediate to specific locations within the nucleolus. We find that early SSU intermediates form clusters near the nuclear envelope that are spatially distinct from later intermediates and LSU intermediates. We are working to discriminate related intermediates at single molecule resolution to map this process within intact nucleoli. These preliminary results show us that 2DTM is a powerful approach to study the spatial organization of ribosome assembly.
Davila et al. (Sun,) studied this question.