Roger M. Burnett

Adenovirus polypeptide IX revealed as capsid cement by difference images from electron microscopy and crystallography

Particles of adenovirus type 2 (ad2), when disassembled, consistently yield groups‐of‐nine (GON) hexons, which are the major virion shell component. The location of a minor component (6%) of the GON has been determined using a novel combination of electron microscopy and X‐ray crystallography. The Brookhaven Scanning Transmission Electron Microscope (STEM) was used to estimate the distribution of protein in the GON to a resolution of 15‐18 A. The relative hexon positions then were determined to within 1 A using a model of the hexon derived from the X‐ray crystal structure to search the STEM image. The difference image between the STEM image and a model hexon group reveals individual monomers of polypeptide IX extending along the hexon‐‐hexon interfaces. The distribution confirms our earlier proposal that four trimers of polypeptide IX are embedded in the large cavities in the upper surface of the GON to cement hexons into a highly‐stable assembly.

The structure of the adenovirus capsid: III. Hexon packing determined from electron micrographs of capsid fragments

The orientation and relative positions of all 240 hexons in the icosahedral outer capsid of adenovirus have been determined. Two types of capsid fragments, obtained after selective disruption of the virion, were analyzed using electron microscopy and image-processing techniques. Planar inverted groups-of-nine, arising from the central region of the capsid facet, were minimally stained to reveal the morphology of restricted regions of their component hexons. Images shown to be related by correspondence analysis were averaged and features of the individual hexon molecule, known from an X-ray crystallographic investigation, were used in their interpretation. The study confirms earlier observations that the hexons in the group-of-nine are distributed on a p3 net, shows that the hexons form a close-packed array using the pseudo-hexagonal shape of the hexon base, and provides their relative positions. Twenty interlocking groups-of-nine account for 180 of the 240 hexons present in the viral capsid. The orientation of the remaining 60 peripentonal hexons was obtained from a rotationally averaged image of a quarter-capsid, a novel viral fragment comprising five complete facets. Each peripentonal hexon forms planar asymmetric interactions with two neighbors in an adjacent group-of-nine so that it lies on an extension of the p3 net. The complete facet thus consists of 12 hexons arranged on a planar p3 net, with a shape that permits interlocking of hexons at the capsid edge. The relative positions of the hexons have been determined to within 5 A using the molecular model, and indicate that the pseudo-hexagonal basal regions are close-packed in a manner that maximizes the hexon-hexon contacts. The results confirm the model proposed earlier for the arrangement of hexons within the adenovirus capsid (Burnett, 1985), and show the power of the inter-disciplinary approach.

Interpretation of electron micrographs of adenovirus hexon arrays using a crystallographic molecular model

Two types of two-dimensional arrays of purified adenovirus type 2 hexon have been obtained and analyzed by Fourier filtration of their electron micrographs. One array contained continuously close-packed hexons, distributed on a hexagonal p3 lattice, with a unit cell dimension of 94 +/- 2 A. The other array contained close-packed hexons with a regular absence, so that rings of six hexons related by sixfold symmetry formed a p6 unit cell. The cell dimension of the hexagonal array was 153 +/- 3 A, with neighboring hexons separated by 88 +/- 2 A. Smaller p6 arrays were also formed by hexons freed from complete virions on the microscope grid by treatment with distilled water. A molecular model of hexon, known from the X-ray crystallographic structure, was used to interpret Fourier-filtered images of the arrays, and to determine the relative orientations of the hexon molecules. The hexon-hexon interaction in the p3 array is that found in the virion facet, whereas that in the p6 array is a planar form of the interaction between peripentonal hexons around the vertex.

Isolation and crystallization of λ exonuclease

Abstract λ Exonuclease is a deoxyribonuclease induced by bacteriophage λ. Mutations in the structural gene for the protein affect general recombination and indicate a possible function for the enzyme. A large scale isolation procedure was employed to purify enough enzyme from a heat-induced λ lysogen for X-ray crystallographic analysis. Analytical ultracentrifugation and SDS-polyacrylamide electrophoresis revealed that λ exonuclease is a tetramer with molecular mass 107,000 Da. Crystallization trials produced morphologically perfect crystals of a size suitable for X-ray diffraction studies. Cubic crystallographic symmetry was indicated by the lack of birefringence when the crystals were inspected with polarized light. X-ray precession photographs indicated that λ exonuclease crystallizes in a space group of P4 1 32, or its enantiomorph P4 3 32, with 24 tetramers in the unit cell of edge 210 A.