Award Abstract #0443899
X-ray Determination of Proteins and Viruses

NSF Org:	MCB Division of Molecular and Cellular Biosciences
Initial Amendment Date:	July 25, 2005
Latest Amendment Date:	April 11, 2008
Award Number:	0443899
Award Instrument:	Continuing grant
Program Manager:	David A. Rockcliffe MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences
Start Date:	July 15, 2005
Expires:	June 30, 2009 (Estimated)
Awarded Amount to Date:	$1110830
Investigator(s):	Michael Rossmann mgr@indiana.bio.purdue.edu (Principal Investigator)
Sponsor:	Purdue University Young Hall West Lafayette, IN 47907 765/494-4600
NSF Program(s):	BIOMOLECULAR SYSTEMS, GENES AND GENOME SYSTEMS
Field Application(s):
Program Reference Code(s):	BIOT, 9183, 7465, 7259, 1166, 1164, 1156
Program Element Code(s):	1144, 1112

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.

PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH

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Choi, K. H., M. C. Morais, D. L. Anderson, M. G. Rossmann.  "Determinants of bacteriophage phi29 head morphology,"  Structure,  v.14,  2006,  p. 1723.

Fokine, A., A. J. Battisti, V. A. Kostyuchenko, L. W. Black, M. G. Rossmann.  "Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses,"  Journal of Structural Biology,  v.154,  2006,  p. 255.

Fokine, A., A. J. Battisti, V. D. Bowman, A. V. Efimov, L. P. Kurochkina, P. R. Chipman, V. V. Mesyanzhinov, M. G. Rossmann.  "CryoEM study of the Pseudomonas bacteriophage phiKZ,"  Structure,  v.15,  2007,  p. 1099.

Fokine, A., P. G. Leiman, M. M. Shneider, B. Ahvazi, K. M. Boeshans, A. C. Steven, L. W. Black, V. V. Mesyanzhinov, M. G. Rossmann.  "Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry,"  Proceedings of the National Academy of Sciences, U.S.,  v.102,  2005,  p. 7163.

Fokine, A., V. A. Kostyuchenko, A. V. Efimov, L. P. Kurochkina, N. N. Sykilinda, J. Robben, G. Volckaert, A. Hoenger, P. R. Chipman, A. J. Battisti, M. G. Rossmann, V. V. Mesyanzhinov.  "A three-dimensional cryo-electron microscopy structure of the bacteriophage phiKZ head,"  Journal of Molecular Biology,  v.352,  2005,  p. 117.

Fokine, A., V. D. Bowman, A. J. Battisti, Q. Li, P. R. Chipman, V. B. Rao, M. G. Rossmann.  "Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins,"  Virology,  v.367,  2007,  p. 422.

Fu, C., M. C. Morais, A. J. Battisti, M. G. Rossmann, P. E. Prevelige, J..  "Molecular dissection of phi29 scaffolding protein function in an in vitro assembly system,"  Journal of Molecular Biology,  v.366,  2007,  p. 1161.

Kanamaru, S., K. Kondabagil, M. G. Rossmann, V. B. Rao.  "The functional domains of bacteriophage T4 terminase,"  Journal of Biological Chemistry,  v.279,  2004,  p. 40795.

Kanamaru, S., Y. Ishiwata, T. Suzuki, M. G. Rossmann, F. Arisaka.  "Control of bacteriophage T4 tail lysozyme activity during the infection process,"  Journal of Molecular Biology,  v.346,  2005,  p. 1013.

Kostyuchenko, V. A., P. R. Chipman, P. G. Leiman, F. Arisaka, V. V. Mesyanzhinov, M. G. Rossmann.  "The tail structure of bacteriophage T4 and its mechanism of contraction,"  Nature Structural and Molecular Biology,  v.12,  2005,  p. 810.

Leiman, P. G., A. J. Battisti, V. D. Bowman, K. Stummeyer, M. Muehlenhoff, R. Gerardy-Schahn, D. Scholl, and I. J. Molineux.  "The structures of bacteriophages K1E and K1-5 explain processive degradation of polysaccharide capsules and evolution of new host specificities,"  Journal of Molecular Biology,  v.371,  2007,  p. 836.

Leiman, P. G., M. M. Shneider, V. V. Mesyanzhinov, M. G. Rossmann.  "Evolution of bacteriophage tails: structure of T4 gene product 10,"  Journal of Molecular Biology,  v.358,  2006,  p. 912.

Leiman, P. G., P. R. Chipman, V. A. Kostyuchenko, V. V. Mesyanzhinov, M. G. Rossmann.  "Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host,"  Cell,  v.118,  2004,  p. 419.

Morais, M. C., K. H. Choi, J. S. Koti, P. R. Chipman, D. L. Anderson, M. G. Rossmann.  "Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29,"  Molecular Cell,  v.18,  2005,  p. 149.

Morais, M. C., M. Fisher, S. Kanamaru, L. Przybyla, J. Burgner, B. A. Fane, M. G. Rossmann.  "Conformational switching by the scaffolding protein D directs the assembly of bacteriophage phiX174,"  Molecular Cell,  v.15,  2004,  p. 991.

Rossmann, M. G., F. Arisaka, A. J. Battisti, V. D. Bowman, P. R. Chipman, A. Fokine, S. Hafenstein, S. Kanamaru, V. A. Kostyuchenko, V. V. Mesyanzhinov, M. M. Shneider, M. C. Morais, P. G. Leiman, L. M. Palermo, C. R. Parrish, C. Xiao.  "From structure of the complex to understanding of the biology,"  Acta Crystallographica Section D,  v.63,  2007,  p. 16.

Rossmann, M. G., M. C. Morais, P. G. Leiman, W. Zhang.  "Combining X-ray crystallography and electron microscopy,"  Structure,  v.13,  2005,  p. 355.

Rossmann, M. G., V. V. Mesyanzhinov, F. Arisaka, P. G. Leiman.  "The bacteriophage T4 DNA injection machine,"  Current Opinion in Structural Biology,  v.14,  2004,  p. 171.

Sun, S., K. Kondabagil, P. M. Gentz, M. G. Rossmann, V. B. Rao.  "The structure of the ATPase that powers DNA packaging into bacteriophage T4 procapsids,"  Molecular Cell,  v.25,  2007,  p. 943.

Xiang, Y., M. C. Morais, A. J. Battisti, S. Grimes, P. J. Jardine, D. L. Anderson, M. G. Rossmann.  "Structural changes of bacteriophage phi29 upon DNA packaging and release,"  The European Molecular Biology Organization Journal,  v.25,  2006,  p. 5229.


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