UCSD Dr. Timothy S. Baker
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Introduction

Macromolecular, cryo-electron microscopy and three-dimensional (3D), image-reconstruction techniques are used in our research to visualize viruses and to determine how they interact with their hosts, replicate and mature. Recent technological advances have led to an explosive growth in this field and have allowed researchers to observe molecules and molecular interactions at sub-nanometer resolutions. Microscopists are now more routinely able to trace protein chains, visualize protein-nucleic acid interactions, and to study how lipids play a major role in some enveloped viruses.

The main benefit of cryo-electron microscopy is that it permits the structures of biological samples to be preserved in a near native state.. To accomplish this, samples are flash-frozen in a sub-millisecond time frame, which prevents ice crystal formation that would damage the sample. Specimen samples are then inserted into a transmission electron microscope and maintained at liquid nitrogen or even liquid helium temperatures without use of any chemical fixatives or stains. UCSD researchers now have access to two, new, state-of-the-art, cryo-electron microscopes. Modern instruments are computer controlled and this greatly assists the operator in day to day tasks. Automated, data-collection software is being used to record the massive amounts of data that are required to obtain high-resolution 3D reconstructions. Automatically-collected data is recorded digitally on CCD cameras and is stored in a database. Data may also be collected on photographic film, an electron detection device that still provides the highest resolution data. A data set of virus images that is large enough to produce a reconstruction to sub-nanometer resolution may contain tens of thousands of individual images.

Computer reconstruction techniques are continuously being developed to more effectively and efficiently extract usable information from the noisy image data. Though many of our computer algorithms exploit the icosahedral symmetry inherent in a large number of the viruses we study, newer programs are being developed to handle images of viruses that don't exhibit such symmetry. Some viruses possess various symmetry-mismatched components and hence must be treated as if they are asymmetric structures. This necessitates the use of many more images in order to achieve a resolution that otherwise could be obtained if the structure were icosahedral. Along with help from San Diego Supercomputer Center staff, we have developed a new system (AUTO3DEM) that automates much of the tedious steps in the 3D reconstruction process and, in favorable instances, enables us to obtain sub-nanometer reconstructions in a manner of days after images have been recorded. AUTO3DEM runs on in-house PC clusters and also on the national Teragrid system.

Current research efforts are focused on structural studies of a wide variety of viral pathogens: these include several fungal partitiviruses; bacteriophage φ29; large, dsDNA viruses that infect algae and insects; reoviruses; adeno-associated viruses; and a family of small, enveloped, ssRNA viruses that include members that are serious human pathogens and have been listed as possible agents of bio-terrorism. Structural studies of the life cycles of these viruses provide basic insights into the mechanisms by which they infect hosts.
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