Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid

Abstract

HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage !, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 A Ê in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 A Ê resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been con®rmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 A Ê . The cell dimensions after post-re®nement were a = 580, b = 626, c = 788 A Ê , = 90.0 . From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even re¯ections along the 0k0 lattice line indicate that the space group is P2 1 . The rotation function was used to determine the orientation of the particles in the unit cell and to con®rm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18 away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at ( 1 4 , 0, 1 4 ) and the other particle at ( 3 4 , 1 2 , 3 4 ). The particle position and orientation were con®rmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 A Ê is under way.

Structure determination of bacteriophage PP7 fromPseudomonas aeruginosa: from poor data to a good map

“…The split peaks also show two extra peaks. The same appearance of this pattern of rotation-function peaks from particles with similar orientations has been observed previously (Oliveira et al, 1993;Muckelbauer et al, 1995;Wikoff et al, 1999) and has been explained as packing peaks (Muckelbauer et al, 1995). Packing peaks can be generated by the symmetry of the packing of particles in the crystal (A Ê kervall et al, 1971), but it is not obvious what features of the packing would generate the observed pattern.…”

The Refined Structure of a Protein Catenane: The HK97 Bacteriophage Capsid at 3.44 Å Resolution

“…10,25,26 The dataset used consisted of 4 797 606 reflections between 190 and 3.44 Å resolution ( Table 4). The merging R-value was 0.160 and the completeness 65%.…”

Increased resolution data from a large unit cell crystal collected at a third-generation synchrotron X-ray source

“…The diffraction and data collection from HK97 Head II crystals to 5 A Ê resolution have been described previously (Wikoff et al, 1998(Wikoff et al, , 1999. Diffraction from the CHESS F1 beamline could be observed on some images to 3.5 A Ê resolution, but the data were very weak and could not be accurately measured.…”

“…We have previously reported the crystallization of HK97 capsids (Wikoff et al, 1998) and preliminary crystallographic results to a resolution of 7 A Ê (Wikoff et al, 1999). The space group was determined to be P2 1 , with unit-cell parameters a = 580, b = 625, c = 790 A Ê , = 90.0 .…”