Double stranded (ds)DNA bacteriophage have been a subject of study for over 60 years and they form the corner stone of modem molecular biology and biophysics. Recently they have been recognized as relevant model systems for herpes virus assembly. The concept of supra-molecular assembly pathways was developed from their intense biophysical study, but not a single high resolution structure has been determined for any of the gene products found in the dsDNA phage particles. Among the most intriguing features common to all dsDNA phage is the maturation of the protein head when DNA is packaged in the preformed procapsid. In this step the lambda- like phage, Hong Kong 97 (HK97), changes diameter from 470 Angstroms to nearly 600 Angstroms, while maintaining exactly the same protein composition. This large scale quaternary reorganization of the subunits is easily visible by electron microscopy and may involve significant refolding of the subunits. We propose to determine the 3.5 Angstrom resolution structure of the mature T=7 (420 subunits) particle of HK 97. The crystalline particles were initially assembled as proheads in E. coli expressing the 42kD viral capsid protein (gene 5) and the viral protease (gene 4). Spontaneous processing of the capsid protein and auto-digestion of the protease produced procapsid II which was then induced to form the mature particle by brief exposure to 6M urea. This material was crystallized and diffraction patterns beyond 4 Angstrom resolution were recorded from orthorhombic crystals with lattice constants a=580, b=627, c=785 Angstroms. A step-wise proposal to obtain the 3.5 Angstrom structure of these particles is presented. Initially the structure at 8 Angstrom resolution will be determined with data currently being collected with a high brilliance, micro focus, rotating anode x-ray source. Initial phases will be determined at 18 Angstrom resolution with a cryo EM structure, in hand, and they will be extended to 8 Angstrom with real space averaging. Data to 3.5 Angstrom resolution will be collected at a synchrotron x-ray source and phases will be extended to this resolution by real space averaging. Of lower priority are proposals to crystallize and determine the prohead structure and to study the prohead to head transition by solution x-ray scattering. These studies will define, at the chemical level, obligate transitions that occur in phage and herpes viridae and that are obvious targets for the development of antiviral agents. Herpes viridae, with diameters in excess of 1200 Angstroms, can not currently be crystallized.
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