Project Summary/Abstract Over the last two decades, many giant DNA viruses have been discovered, some of which are bigger than a small cell. How these giant viruses assemble their virion shell from thousands of simple protein building blocks so precisely is a mystery. However, the sheer size of these viruses poses a significant challenge to currently available techniques. This project will tackle this challenge by using a marine giant virus Cafeteria roenbergensis virus (CroV) as a model to decipher the assembly mechanism of giant viruses, and as an opportunity to develop technology to push the resolution limit of these gigantic structures to the atomic level. During the last five years, cryo-electron microscopy (cryo-EM) has become an increasingly powerful tool to study the structures of biological molecules at atomic resolution, earning its developers the 2017 Nobel Prize in Chemistry. We will collect higher quality images using state-of-the-art cryo-EM equipped with latest new hardware, such as energy filters, direct electron detectors and phase plates. Using these images together with new software algorithms, we will determine the structure of giant CroV to high resolution by image analyses and reconstruction. Structures of individual CroV proteins will also be solved to atomic resolution by cryo-EM using various methods and docked into the cryo-EM reconstructed maps. The resultant pseudo-atomic structure will allow characterization of the ultrastructural features and architecture of CroV, building the essential foundation to unravel the assembly of giant viruses. The structure information will be combined with classic biophysical, molecular dynamic simulation, mathematical modeling, and computational analyses to evaluate the novel assembly model of giant viruses. In the new assembly model, the protein shells of giant viruses are assembled continuously from the 5-fold vertices in an interesting spiral way instead of assembled from patches in a step-wise fashion previously assumed. Giant virus protein shell is assembled from protein building block similar to other viruses, including many human pathogens. Some giant viruses have been associated with human diseases such as pneumonia and cognitive functional change. Understanding these principles governing the assembly of giant viruses will improve the development of therapeutic agents to inhibit virus assembly, thus providing a new avenue for preventing and treating viral diseases in general. Elucidation of the molecular interactions that drive assembly of these giant viruses will also shed light on how to control protein-protein interactions effectively, facilitating the rational design of virus-like nanoparticles with a wide size range for biomedical and other nano-applications. Since some giant viruses are bigger than a small cell, techniques and methods developed in this project will push the limits of structural biology and provide new and useful tools to study even larger supramolecular assemblies and eventually the whole cell in the future.