Double stranded (ds) DNA bacteriophage particles are accessible """"""""molecular machines"""""""" that often contain 20 or more gene products that control assembly, maturation, DNA packaging and storage, cell attachment and DNA delivery. A number of these phages, including T7, T4, P22, phi29, lambda, and their close relatives, are among the best-characterized viruses at the genetic and phenotypic levels. These systems provide many features that are common in biology, but they can be more readily isolated, investigated, and physically and genetically manipulated than many cellular components of comparable size. What's more, these particles possess many of the characteristics envisioned for designed nano-structures and may serve as the basis for their development. Until recently the mechanistic characterization of the particle functions of these viruses has been hampered by the lack of high-resolution structures of any of the proteins residing transiently or permanently in the particles. During the last funding period, support from this grant lead to an atomic model of the lambda-like HK97 recombinant, mature capsid based on a 3.6A resolution electron density map (space group P2, a=580, b=625, c=790A, p=90.0o). The structure explains the remarkable physical (chain mail) cross-linking of hexamers and pentamers in the T=7l surface lattice and has suggested numerous mutations to define the mechanisms of particle expansion and autocatalytic ligation of ASN and LYS side chains. We now propose to empirically define the structural pathway followed during the expansion from prohead II (diameter=450A) to head I (diameter 650A) by determining the high-resolution structure of prohead II. Crystals of this intermediate currently diffract to 6A and a l0A data set was collected, analyzed and particle orientations and positions determined in the P2,3 (a=704A) space group. Models will be fitted to 20A-electron density of intermediates in the expansion determined by time resolved cryo-EM and image reconstruction by our collaborators Alasdair Steven and James Conway. Crystallographic studies of HK97 prohead I, containing a virally encoded protease and full-length capsid protein, as well as mutant capsid protein assembled as hexamers but incapable of assembly into particles, will be performed with material supplied by our collaborators Roger Hendrix and Robert Duda. The long-term goal is to define the mechanism of the coordinated subunit tertiary and quaternary interactions that lead to a particle expansion from 450A to 650A, while maintaining the identical protein composition.
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