T7 is one of the most studied bacteriophage systems for which a wealth of genetic and biochemical information is known. Similar to other tailed dsDNA phages, the morphogenesis pathway of T7 starts with a DNA-free empty procapsid that is formed through scaffolding protein-assisted assembly. The procapsid undergoes large-scale conformational changes and expands into the capsid II state during the maturation process, which is triggered by DNA packaging through the portal vertex. An infectious particle is formed only after the completion of DNA packaging and tail assembly on the portal vertex. Although phage morphogenesis has been extensively studied for numerous phage systems, including T7, many questions remain to be answered to understand its mechanism. Having been able to obtain some preliminary results on the structures of the T7 capsids, we propose to carry out more systematic studies to elucidate the structural basis of T7 assembly, maturation and DNA packaging.
We aim to solve the structures of the capsid shell, the scaffolding proteins, the portal complex, the internal cylindrical core, the tail and the DNA packing. These structures will be examined at each of their capsid states in the morphogenesis pathway to probe their roles in the maturation process. State-of-the-art cryo-electron microscopes and computer 3-D reconstruction techniques will be used to accomplish these tasks. We will use the popular image processing software EMAN, of which the PI is a co-developer, to determine the capsid shell structures to 7-8 A or better resolution by imposing icosahedral symmetry. We will also further develop the image processing algorithms to push for 4-5 A resolution structures for the infectious phage particles. Our recent extension of EMAN to allow the solution of structures of symmetry matches will be used to determine the structures of the scaffolding proteins, portal complex, cylindrical core and tail in the intact capsids at 15 A resolution or better. These proteins do not have icosahedral symmetry and cannot be resolved using classic reconstruction methods that rely on icosahedral averaging. This method will be further developed to allow direct tracing of the dsDNA strands and visualization of dsDNA packing. The T7 structures will form the foundation for understanding the basic processes of assembly, maturation and genome packaging of not only the tailed dsDNA phages, but also the related human herpes viruses. ? ? ?
