X-ray Determination of Proteins and Viruses

Purdue University, West Lafayette IN

Investigators

Abstract

In this project, X-ray diffraction and cryo-electron microscopy (cryoEM) techniques, in conjunction with molecular biology and protein chemistry, will be the primary tools used to analyze the life cycle of various bacterial viruses. The emphasis will be on the small and simple double-stranded DNA (dsDNA) tailed bacteriophage phi29, on the large and complex dsDNA bacteriophages T4 and even larger phiKZ, as well as on the tailless icosahedral ssDNA bacteriophage phiX174. The objectives will be to determine not only the structures, but also the molecular mechanisms by which the assembly intermediates are stabilized, how the DNA is packaged into the empty pre-assembled proheads, how each step triggers the next during viral morphogenesis, and to study the huge conformational changes that occur when the virus infects a host cell. Comparison of bacterial and animal viral systems is likely to recognize common strategies and evolutionary origins that are not as easily determined by the study of animal viruses alone. Many bacterial viruses are too awkwardly shaped to achieve a packing organization that is compact enough, with sufficient interparticle contacts, to make crystallization feasible. In addition, these viruses have numerous fibrous sensor attachments that are flexible and variable in orientation that would make crystallization impossible. Although cryoEM investigations of viruses seldom extend beyond 10-angstrom resolution at this time, these studies can be augmented by separately studying the crystal structures of the viral components expressed in suitable prokaryotic or eukaryotic systems. A pseudo-atomic resolution structure can then be obtained by fitting the individual crystal structures into the cryoEM images of the virion, assembly intermediates, or larger fragments of the native virus. Broader Impacts: The project will involve undergraduates, graduate students, and postdoctoral fellows. The PI has been active in mentoring women as well as underrepresented minority researchers. In addition, the research will be integrated in the teaching activities, including a course on crystallography. As in the past, the project will involve development of new techniques, such as combining electron microscopy and crystallography. Such techniques will be useful to the scientific community working on the structural biology of large complexes.