Tim Baker¹, and Tom Goddard²
¹ University of California, San Diego
Resource for Biocomputing, Visualization, and Informatics
University of California, San Francisco
Our research focuses on applying electron cryo-microscopy (cryoEM) and three-dimensional (3D) image reconstruction techniques to examine the structures of large macromolecular complexes at sub-nanometer resolutions. We use intermediate voltage, state of the art transmission electron microscopes to image vitrified biological samples (primarily viruses) in near native, fully hydrated conditions. By collecting thousands and sometimes tens of thousands of individual particle images, we are able with software developed in-house to reconstruct the 3D structures of many different viruses. These structures, in concert with correlative data from biochemical experiments or other structural techniques such as X-ray crystallography, provide clues to understanding how viruses function. That is, how does virus structure provide the means for viruses to recognize, bind, and infect cells?
Our studies employ a variety of visualization tools to aid in analyzing and interpreting complex macromolecular structural data. Among the tools available (including some developed by us), Chimera has rapidly become the de facto visualization platform adopted by our lab because it offers a rich exploration and analysis environment suitable for our particular needs. Of course, as is true with virtually any similar system, functionality has limits. We are therefore excited about the prospects of working closely with members of the Chimera development team to enhance and expand the capabilities of Chimera to help researchers like ourselves develop new and efficient ways to visualize complex structures. The following sections provide brief glimpses of some of the structural investigations currently underway, with a few illustrations of results displayed using Chimera.
Characterization of the capsid organization in large viruses
The capsids of large viruses (diameters > 100 nm) contain hundreds or thousands of subunits organized in very precise arrangements. We have used cryoEM to visualize directly the quasi-symmetry and capsomer organization in several very large (> 180nm diameter) dsDNA viruses. As one example, chilo iridescent virus (CIV), the type species of the genus Iridovirus (Family Iridoviridae), has a maximum diameter of 1850Å. Our unpublished 3D image reconstruction of CIV at 13Å resolution  reveals a protein shell consisting of 12 pentavalent (pentamer) and 1460 hexavalent (trimer) capsomers, arranged with T=147 (h = 7, k = 7) icosahedral, quasi-equivalent symmetry. Previously, numerous visualization tools were unable to effectively handle the large size (in pixels) of the CIV 3D map. Modifications to Chimera have since made it possible to load the entire 3D volume for interactive rendering and this has permitted us to illustrate in a variety of ways the complex organization of the CIV capsid (see image below). With an appropriate coloring scheme it is easy to illustrate where the 12 pentasymmetrons and 20 trisymmetrons lie in the icosahedral surface lattice. Each pentasymmetron is composed of 30 trimers (blue) and one pentamer (red). Each trisymmetron is composed of 55 trimers, among which 28 trimers are located on the edge of the symmetron (pink) and 27 are located inside the symmetron (green). An additional Chimera module was designed to help quantify the degree to which the capsid deviates from a perfect sphere or a perfect icosahedron. Such data are useful for better understanding the underlying principles of assembly in such large viruses.
Assembly and maturation of Nudaurelia capensis ω virus
Nudaurelia capensis ω virus (NωV) is a T=4, icosahedral insect virus with a bi-partite, positive sense, RNA genome. Structural studies of the procapsid have shown the quasi-equivalence among all subunits. The similarity of the subunits and their interfaces demonstrates that the large, porous procapsid is an assembly intermediate, which just precedes capsid maturation. Comparison of the capsid stuied by cryoEM at pH 5 and the crystal structure studied at pH 7 shows that they share the same structure and demonstrates that the mature capsid is a stable structure, which doesn't alter between these two pH extremes. Maturation of NωV involves an autocatalysis of the capsid subunit. A point mutation at the cleavage site abolishes maturation cleavage of virions and leads to a reversible conformational change that allows the large procapsid to transform into a small capsid on going from pH 7.6 to pH 5. Detailed comparisons between reconstructions exhibiting cleavage or not have been examined using Chimera tools (see image below). This has revealed some clues regarding why maturation cleavage leads to a stabile capsid and prevents reversible conformational change .
High throughput image reconstruction
Considerable efforts are directed at coupling automatic data collection and image processing strategies on high throughput computer clusters . The goal is to enable us to study a wide variety of complex macromolecular machines at the highest possible resolutions (better than 5Å) and in the shortest possible time (realistically in man-days instead of man-years). The challenges this poses are of course quite significant, yet we fully recognize the need to continue to integrate the production of reliable 3D data coupled to rigorous visualization and analysis tools like Chimera.
- Yan X., Chipman P.R., Battisti A.J., Bergoin M., Rossmann M.G. and Baker T.S. (2005). The Structure of the T=147 Iridovirus, CIV, at 13A Resolution. Proceedings of Microscopy and Microanalysis, 11(S02): 134-135.
- Tang J., Chipman P.R., Zhang W. and Baker T.S. Cooperative interactions between the glycoprotein and nucleocapsid protein in sindbis virus budding. In preparation.
- Tang J., Olson N., Anderson D. and Baker T.S. CryoEM reconstruction of the phi29 shows the unaveraged structure of the DNA packaged machine. In preparation.
- Tang J., Lee K.K., Bothner B., Baker T.S., Yeager M. and Johnson J.E. Multiple conformational states of a virus capsid imaged at subnanometer resolution: implications for capsid assembly and maturation. In preparation.
- Zhang X., Ji Y., Zhang L., Harrison S.C., Marinescu D.C., Nibert M.L., Baker T.S. (2005). Features of reovirus outer capsid protein mu1 revealed by electron cryomicroscopy and image reconstruction of the virion at 7.0 Angstrom resolution. Structure, 13(10):1545-57.
- Yan, X., Sinkovits, R.S., Baker, T.S. (2006). AUTO3DEM - an automated and high throughput program for 3D image reconstruction of icosahedral particles. J. Struct. Biol., submitted.
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