The original 1963 title of this project, "X-ray Determinations of Proteins and Viruses," is still apt today. In the 60s and 70s the project supported primarily the development of technology for structural studies. However, starting in the 90s, with the successful determination of the first virus structures and the establishment of crystallography as an automated tool, this project was directed to support the study of viruses that infect and can kill bacteria ("bacteriophages"). In the period of the current project there is a shift back to include not only analyses of bacteriophages, but also the development of new tools for the analysis of pleomorphic viruses by means of cryo-electron tomography (cryoET). X-ray diffraction and cryo-electron microscopy (cryoEM) techniques, in conjunction with molecular biology and protein chemistry, will be the primary tools used to analyze the life cycle of various bacterial viruses. The emphasis will be on the small, single-stranded DNA, tailless bacteriophage phi X174; on the double-stranded DNA (dsDNA), short-tailed phage C1; on the small, dsDNA, tailed phage phi 29; as well as on the large and complex dsDNA phage phi KZ. Part of the emphasis of this project will be to extend some of the previous cryoEM reconstructions to make possible visualization of individual amino acids that determine chemistry and, hence, the function of the viral components. In addition, studies have been initiated on the interpretation of cryo-tomograms of lipid enveloped viruses that do not obey exact icosahedral symmetry. Such viruses are either too awkwardly shaped to achieve a packing organization that has sufficient inter-particle contacts to make crystallization feasible, or are pleomorphic (many different shapes) with no virion having exactly the same shape or form. Where necessary, cryoEM and cryoET will be augmented by crystal structures of viral protein components. Pseudo-atomic resolution structures can be obtained by fitting the individual crystal structures into the cryoEM or cryoET images of the virion, assembly intermediates or larger fragments of the native virus. By combining structural results with mutational and other studies of assembly intermediates, genome packaging and host recognition, the hope is to probe the mechanisms of these biological processes. As functions such as assembly and host recognition that are required by both bacteriophages as well as by viruses, information gained in the study of bacteriophages can be helpful in the study of all viruses.
Broader Impact
The co-authors of the papers published over the previous five years of NSF support for this project include one undergraduate, three graduate students, eleven post-docs and three technicians. Of these, seven were women and their ethnic backgrounds included one Hispanic, three Chinese, one Japanese, three Russian, one Korean, one Canadian and four US Americans. All three of the graduate students have now received their Ph.D. degrees and went on to do postdoctoral training. Two of these students are now assistant professors at major universities. All students and most post-docs took advantage of a semester long courses in crystallography (taught by the PI) and electron microscopy. Similar statistics are likely for the next five years of this project. Since 1963 more than 130 post-docs and 30 graduate students have benefited from the continuing NSF support. Many of these students and post-docs have subsequently become internationally recognized structural biologists.
