Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is si milar in architecture to bacteriophage lambda, the type member of this fami ly, with an icosahedral capsid of triangulation number T = 7. No high-resol ution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-r ays. At 650 Angstrom in diameter, the large size of the particle and result ant large unit cell create crystallographic challenges. The empty Head II ( mature) particles were expressed in Escherichia coil and assembled in vitro , but they have the same morphology as the mature HK97 capsid. Previously r eported Head II crystals diffracting to 3.5 Angstrom resolution are examine d here in detail. Although the cell dimensions suggest an orthorhombic latt ice, further analysis demonstrated that the space group was monoclinic. Thi s has been confirmed by the present study. Images were recorded on the Fl b eamline at CHESS and they were processed and scaled, resulting in a data se t with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 Angstrom. The cell dimensions after post-refinement were a = 580, b = 626, c = 788 Angstrom, beta = 90.0 degrees. From the particle dimensions determ ined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even reflections along the 0k0 lattice line indicate that the space group is P2(1). The rotation func tion was used to determine the orientation of the particles in the unit cel l and to confirm the space group. An icosahedral twofold axis is approximat ely, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to th e b axis, is rotated 18 degrees away from the a axis. The centers of the tw o particles must be positioned close to the minimum-energy packing arrangem ent for spheres, which places one particle at (1/4, 0, 1/4) and the other p article at (3/4, 1/2, 3/4). The particle position and orientation were conf irmed by calculating a Patterson function. The particles interact closely a long icosahedral threefold axes, which occurs both along the crystallograph ic a axis and along the b axis. The particle dimensions derived from this p acking arrangement agree well with those determined by cryo-EM and image re construction. The cryo-EM reconstruction will be used as a model to initiat e phase determination; structure determination at 7 Angstrom is under way.