Bacteriophages have long been used as model systems for the study of gene expression, macromolecular assembly, and for cloning vectors in genetic engineering. Bacteriophage P22 is among the most comprehensively studied bacteriophages using biochemical and genetic tools. The availability of numerous P22 mutant capsids and assembly intermediates makes it possible to identify key proteins within the capsids and to study assembly pathways and mechanisms. Based on our previous success on low resolution structural studies of P22 capsids in different functional and chemical states in collaboration with Jonathan King and Peter Prevelige, we propose to embark on a more systematic and co-ordinated effort to further characterize the structural basis of the P22 morphogenesis. In this proposal, we specifically aim at understanding the structural mechanisms for scaffolding protein assisted procapsid assembly, capsid shell expansion upon maturation, and the modes of internal DNA organization and encapsidation. To achieve this, we will employ electron cryomicroscopy and computer reconstruction as the structural tools to study a variety of P22 assembly intermediates and chemically or genetically modified particles. We will target the structural determinations to approximately 7 Angstrom units resolution using Fourier Bessel reconstruction algorithm for particles with icosahedral symmetry. We also plan to develop algorithms to determine the structure of the portal protein complex within the capsid through a hybridized image processing tools aimed at understanding the mismatched symmetry between the non-icosahedrally organized DNA entry port and the icosahedrally packed coat proteins.
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