Structural Biology Laboratory

Structural Studies of Filamentous Biological Assemblies

Gerald Stubbs, Professor of Biological Sciences

We use fiber diffraction, protein crystallography and electron microsopy to look at disordered and partially ordered filamentous biological assemblies.

Most of our research interests fall into one of three areas:

Structures of filamentous viruses

Structures of amyloids, the mis-folded proteins found in Alzheimer's, BSE ("mad cow disease") and other neurodegenerative diseases.

Development of new methods in fiber diffraction

Fiber Diffraction Workshop, August 6 - 9, 2006, Fall Creek Falls State Park

Virus Pictures	Laboratory Publications	Trip to the APS, August 2001	Biological Sciences Home Page
RCN: FiberNet	BioCAT	SSRL Beamline 4-2	Prusiner Laboratory

Why study plant viruses?

Basic virology. We are interested in viral structure and function at the most fundamental mechanistic level. Assembly, disassembly, and host-viral interactions are all fundamentally biochemical processes; neither understanding nor intervention is possible without structural studies. More complex animal viruses have been studied structurally, but virology still needs to push simple model systems to the limit, as well as investigating more complex systems in less depth. Our old workhorse, tobacco mosaic virus, is without doubt the best understood of all viruses, and has been the model system for almost every fundamental advance in virology in the last hundred years.

Biotechnology. Tobacco mosaic virus and potato virus X are being used by our collaborators to express a variety of pharmacologically important peptides, functioning as antibiotics, vaccines, and other useful molecules. Structural studies are directed at understanding and overcoming the reasons why such constructs fail to assemble or disassemble, or aggregate - the answers are all in the structures!

Agriculture. Although our laboratory is primarily interested in basic research, we are always glad if anything we come up with is useful to agriculture. We hold a patent on a type of transgenic plant that resists viral infection to a much greater degree than any previously known.

Why use fiber diffraction?

Structural biology has generally concentrated on compact, ordered molecules and assemblies, simply because they are much more tractable, and can be studied, for example, by the highly successful and now almost routine methods of protein crystallography. But many biological polymers and assemblies are long, helical, filamentous structures. Examples include the filamentous viruses, cytoskeletal filaments, bacterial flagellae, chromatin, components of the extra-cellular matrix, and many simple polymers such as nucleic acids and polysaccharides. The components of these assemblies are difficult to crystallize, since their natural tendency is to form filaments. Even when they do crystallize, the molecular interactions in the crystals rarely correspond to the biologically significant interactions in the fibers. Fiber diffraction is the only practical method of structure determination at the molecular level for these assemblies.

The difference between fibers and crystals is that, in fibers, the molecules are parallel to each other (as they are in crystals), but they are randomly rotated about the long axis of the fiber. As a result, the data obtained from an x-ray fiber diffraction experiment are cylindrically averaged relative to the data that would be obtained from a hypothetical crystal of the same aggregate. Information is lost, but often not so much that the structure can not be determined. Much of the effort of this laboratory over the years has been directed toward obtaining structures from fibers comparable to those obtained from good crystals of similarly large assemblies.

Projects in this laboratory

We are working with a wide variety of filamentous plant viruses, and projects range from viruses new to this laboratory, where the focus is still on production and purification, to the venerable old tobacco mosaic virus (this project was 100 years old in 1998!). Most of the TMV work is now collaborative with other laboratories who are applying our structural results to such areas as biotechnology and crop protection. Among our other projects, the potexviruses and potyviruses are both important to agriculture and biotechnology; potato virus X has great potential in biotechnology, and the potyviruses (which some people have estimated to make up one third of all plant viruses) are responsible for half the viral crop damage in the world. Some of the viruses we are working with include:

Potato virus X

This virus is important as a model for filamentous structures and more complex viruses and as a vector in biotechnology. It is one of three plant viruses that have been used successfully to produce pharmacologically useful peptides attached to their coat proteins. Peptides expressed in this way are unusually easy to produce and purify in large quantities, making use of the high titers and ease of purification of the viruses themselves. PVX is also responsible for the destruction of about 10% of the world's potato crop.
We are working with both intact virions and the isolated coat protein. We have obtained excellent fiber diffraction patterns from the virions, and small but promising crystals of the coat protein.
Papaya mosaic virus

This is a potexvirus, closely related to potato virus X. A structure determined for PMV would be a reasonable model for the PVX structure. PMV is easily grown in papaya trees, and it is available in exceptionally large quantities (we can obtain hundreds of milligrams in a single purification. For these reasons, more is known about its properities (including the nature of its assembly and disassembly) than about any other potexvirus. We are interested in PMV is as a model for other potexviruses, many of which are of great importance in virology, agriculture, and biotechnology.
Narcissus mosaic virus

Another potexvirus, interesting because of a variety of spectroscopic and structural studies that have been carried out by other groups.
Potato virus Y

Potato virus Y is the type member ("official" typical example) of the Potyviridae family. Although potyviruses are immensely important to agriculture and have biotechnological potential, almost nothing is known about them at the level of molecular structure.
Bean common mosaic necrosis virus

Another potyvirus. Potyviruses are difficult to work with, because they tend to aggregate in solution, they are easily degraded by naturally occuring plant proteases, and they are difficult to produce in high yield. BCMNV is more stable than most, and is obtainable in slightly higher yields.
Wheat streak mosaic virus

A potyvirus. More soluble than other potyviruses, so this one may be a better candidate for making oriented sols for fiber diffraction and coat protein crystals for X-ray crystallography.
Sunnhemp mosaic virus
Odontoglossum ringspot virus

These last two are tobamoviruses, and have been widely used by molecular virologists to study host specificity. Why does a virus attack one species and not another? At least part of the answer seems to lie in the interactions between the coat protein and some of the host proteins, so coat protein structure determination is an obviously useful approach to this question.

We are happy to provide samples of our viruses to other laboratories. A few milligrams, particularly of TMV, can usually be provided on request; larger quantities may require special arrangements. Recipients are responsible for arranging any necessary permits etc.

Work in this laboratory is supported by National Science Foundation Grant MCB-0743931, National Institutes of Health Grant P01 AG010770, and by a Discovery Grant from Vanderbilt University. Any opinions, findings, and conclusions or recommendations expressed in this web site are those of the author and do not necessarily reflect the views of the National Science Foundation or the United States Department of Agriculture.

Students Andrew Wilson, Don Becker, and Zurabi Lominadze work on a helium tunnel during a trip to BioCAT in October 2006.

Contact Information

Gerald Stubbs, Professor of Biological Sciences

Why study plant viruses?

Why use fiber diffraction?

Projects in this laboratory

Potato virus X

Papaya mosaic virus

Narcissus mosaic virus

Potato virus Y

Bean common mosaic necrosis virus

Wheat streak mosaic virus

Sunnhemp mosaic virus

Odontoglossum ringspot virus