Our laboratory has demonstrated the capability of solving medium resolution (7-9 Angstroms) structures of several icosahedral particles by combining several thousand-particle images recorded in an electron cryomicroscope. At these resolutions, we have been able to identify most of the alpha helices and beta sheets of the proteins. Presently, the process of data collection and computer image reconstruction is very labor intensive, even for experienced users, and has a very steep learning curve for new users. We propose to develop a high throughput procedure so that the process of structure determination of icosahedral particles at medium resolution - from data collection to structure determination and interpretation - can be completed within 3-5 weeks. This represents a factor of 10-20x reduction in time for completion of each project. This technological advance will allow molecular virologists to use such structures as a standard approach to understand structure and function relationship in the context of virus assembly and interactions with cellular factors such as antibodies and receptors. This proposal consists of two developmental components. The first is to develop semi-automatic on-line digital image acquisition with a 4k x 4k CCD camera in our newly acquired 200 kV electron cryomicroscope with a field emission gun. The second is to improve the computational efficiency of existing software and to integrate much of the stand-alone software, which includes particle picking, microscope parameter determination, particle orientation and center refinement, 3D reconstruction and structural interpretation. We will use the Python scripting language to coordinate these programs and use an object-oriented database to manage the metadata throughout the project data acquisition and analysis. This software will be made freely available to the research community. We will deploy these technology enhancements to our already funded projects on P22 bacteriophage and herpes virus in addition to a new project with the Venezuelan Equine Encephalitis virus, a select agent with potential bioterrorism threat.
| Booth, Christopher R; Jiang, Wen; Baker, Matthew L et al. (2004) A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope. J Struct Biol 147:116-27 |
| Jiang, Wen; Baker, Matthew L; Wu, Qiu et al. (2003) Applications of a bilateral denoising filter in biological electron microscopy. J Struct Biol 144:114-22 |
