MICROSCOPY & IMAGE ANALYSIS OF UNSTAINED MACROMOLECULES

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The proposed studies involve structural analyses by cryoelectron microscopy and three-dimensional image analysis of a wide range of viruses. Our immediate goals are to answer fundamental questions about the morphology and composition of complex biomacromolecules. Along with knowledge gained from biochemical, genetic, immunological, and other biophysical studies, our structural work can provide new insights about the assembly, stability, and other functional properties of macromolecules. Our aim continues to focus mainly on structural studies of macromolecules that are too large or too complex to examine with current crystallographic technology. The extensive number and scope of our studies is made possible through several fruitful collaborations. The three-dimensional structures of a wide variety of icosahedral viruses, subviral particles, and virus-antibody complexes will be examined. These include members of the Papovaviridae (polyoma virus, simian virus 40, and bovine, human, and cottontail rabbit papilloma viruses), the Reoviridae (rhesus rotavirus and serotype-1 reovirus), the Comoviridae (cowpea mosaic virus [CPMV]), the Tetraviridae (Nudaurelia capensis beta and omega viruses), and isometric prohead mutants of the Bacillus subtilis bacteriophage phi29. Methods to study the morphology of the native (non- isometric phi29 head will be investigated. These viruses are all widely studied under a diverse range of biological disciplines. Despite an immense wealth of information, detailed structural knowledge about many of these viruses is limited. The organization of protein and nucleic acid components in each of the molecules will be studied, as well as the protein-protein and protein- nucleic acid interactions responsible for the stability, assembly, and disassembly of the complexes. The stoichiometries and relative distributions of polypeptides in these macro-molecules will be examined by antibody labeling and by comparing the structures of mature particles (e.g. full virions) with partially assembled or disassembled particles (e.g. intermediate subviral particles, core particles, empty capsids, etc.). Knowledge of the atomic structure of CPMV will guide our examination of the mechanism of virus neutralization in the cryo-electron microscopy studies of complexes of CPMV with Fab and IgG molecules. The methods of cryoelectron microscopy and image analysis will be further developed i) to allow the structures of large and highly complex molecules to be studied, ii) to make processing procedures more flexible and robust as well as efficient, iii) to map virus neutralization sites with antibody and antibody-fragment labels, iv) to examine nucleic acid and protein- nucleic acid organization inside virions, v) to examine the effects of electron radiation damage on frozen-hydrated specimens, vi) to improve contrast and resolution in reconstructed density maps (e.g. by correcting for phase contrast transfer effects or by averaging very large numbers of particle images), and vii) to phase x-ray crystallographic data with models derived from density maps reconstructed from micrographs.