Instrumentation for upgrading cryoEM and single particle analysis capabilities Abstract Examination of the 3 dimensional structure of large macromolecular assemblies often of megadalton mass or greater remains beyond the reach of X ray or NMR methods. Transmission electron microscopy however provides a means of directly visualizing large protein and nucleic acid-protein assemblies. Newer approaches in which images of hundreds to thousands of individual structures are digitized and processed using computer based averaging routines provide a means of generating 3-D structures with resolution in the range of 2.0 nm when samples are imaged by negative staining. Further structural detail can often be obtained by imaging the samples frozen in ice using cryoEM methods and this can increase the resolution into the range of 1.0 nm. The basic skill for carrying out these methods are in place at the University of North Carolina Medical School and both 3-D averaging of negative stained images and cryoEM preparation of samples has been carried out. To advance the studies of a group of active NIH funded investigators, several critical pieces of equipment are needed so that state of the art work in this area can be carried out. A key item is a Vitrobot computer controlled robotic freezing instrument which will make it possible to freeze sets of grids all with the same characteristics for transport to a higher voltage FEG equipped microscope until such an instrument is available in the Research Triangle area. In addition to this instrument, a new cryotransfer system is requested as well as two new cameras of the 120 kV TEM which are needed for collection of large numbers of images for processing. The research projects which will utilize this instrumentation include studies of DNA replication complexes (Griffith), DNA repair complexes (Ramsden and Sancar), protein and DNA-protein filaments formed by Herpes virus proteins (Griffith, Damania, Raab-Traub), Coronin 1B protein filaments in the cell (Bear), membrane bound oncogenic proteins (Raab-Traub), protein filaments related to neurodegenerative diseases (Dokholyan), protein structures in the eye (Costell), and new engineered viruses being developed as vaccines against HIV (Johnston). The instrumentation will be housed in the Lineberger Comprehensive Cancer Center EM core facility and its administration, upkeep, and training in its use will be the responsibility of this facility which has been in operation for 27 years. Public Health Relevance: Instrumentation for upgrading cryoEM and single particle analysis capabilities Narrative The requested equipment will expand research efforts for multiple investigators at the University of North Carolina at Chapel Hill. These investigators are working to understand the cellular mechanisms involved in Cancer, HIV vaccines and Ophthalmologic and Neurodegenerative diseases. This understanding can then be translated into patient treatment.
| Remus, Dirk; Beuron, Fabienne; Tolun, Gökhan et al. (2009) Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139:719-30 |
