The Imaging Sciences Laboratory has a major collaborative research effort with the Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs and NMR spectra with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids. Ongoing research involves analyses of structures related to herpesvirus and papillomairus as well as other icosahedral virus capsids. Papillomaviruses (e.g. HPV-16) encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein L2 is not required for capsid formation, it is thought to participate in encapsidation of the viral genome, and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. Cryo-electron microscopy and difference 3D reconstruction analysis of purified capsids revealed an icosahedrally-ordered L2-specific density beneath the axial lumen of each L1 capsomer. In addition, we have studied three biochemical constructs to try to determine how much L2 is contained within HPV-16 capsids. These results were published in J. Virology (2008). We have used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV-16 capsids assembled in 293T cells. The major capsid protein, L1, initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter;its pentameric capsomers change in structure, most markedly in their axial region;and the interaction surfaces between adjacent capsomers are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. The C175S mutant, which does not crosslink, shows similar maturation-related structural changes but capsids are significantly larger, under otherwise similar conditions. We conclude that the observed structural size changes facilitates maturation, but crosslink formation is required to lock the capsid into the mature state. These results will submitted for publication. We have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using NMR data. Development of Xplor-NIH has continued in the following areas: (a)further development of the Python scripting interface along with extensive documentation;(b) the ability to refine against fiber X-ray diffraction data;(c) additional tools and facilities for refining molecular structures directly against solution X-ray scattering and small-angle neutron scattering data;(d) the PASD facility for automated Nuclear Overhauser Effect peak assignment has been substantially improved;(e) a new structure refinement target has been introduced such that calculated protein structures have the correct molecular density.
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