While a recent ?resolution revolution? has catapulted the use of cryo-EM imaging for high-resolution structure solution of biomolecules, these advances have mostly been restricted to studies of relatively large complexes (e.g. ribosome, spliceosome, and viral particles). Properties of these complexes, such as having a molecular weight >200 kD and inherent stability, make them ideal targets for cryo-EM, which utilizes the averaging of data from tens/hundreds of thousands of particles to reconstruct their 3D structure. However, the vast majority of molecules-of-interest do not have these properties, and have thus remained ignored and considered intractable for cryo-EM studies. Here, design and initial development of four technologies that can overcome these hurdles to make the cryo- EM imaging practical to the research and biomedical communities are described. First, inspiration has been taken from viral particles to produce a rigid and versatile scaffold for the capture and presentation of proteins via a ?plug-and-play? approach. Because this approach does not require genetic fusion of each new protein-of- interest to the scaffold, ?average-sized? proteins and their variants can be rapidly screened for structural study. Here these technologies will be advanced using molecules from the PIs' field of expertise (protein/RNA biology), but these scaffolds and methods can be applied to any functional class of proteins-of-interest. Second, a rigid and versatile scaffold for the capture and presentation of nucleic acids has been produced. Technologies based upon this scaffold will open the doors to cryo-EM imaging of folded RNAs that have been heretofore considered too small for cryo-EM and transmission EM (TEM) studies. Moreover, structural studies and measurements of the in vivo variance of RNA folding populations in cells will be determined using these technologies. In this proposed work, technologies will be developed for wide scale use by the community by determining and optimizing the parameters that affect their use in cryo-EM and TEM studies of proteins and RNA. Ultimately, these rigid, highly symmetrical, and versatile scaffolds, and the methodologies for their optimal use, will drive down the lower practical size limits of cryo-EM and enable researchers to address a wide range of biological questions specific to their respective fields.
This work will develop and advance scaffold and capture technologies that will make the astonishing advancements in cryogenic electron microscopy (cryo-EM) accessible to many more investigators. The technologies proposed aim to reduce the practical lower size limit to ?average-sized? biopolymers, instead of only larger ones, thereby allowing full deployment of cryo-EM. Here, we focus on a diverse set of proteins and RNAs as proof-of-concept molecules owing to their chief importance to myriad cellular functions and disease.
