The purpose of this grant request is to obtain equipment for adapting our high resolution JEOL 2000 electron microscopy for cryo-imaging and diffraction. The requested equipment includes cold stages, an anticontaminator, a TV-rate camera, slow-scan CCD camera and accessory equipment for maintenance of the cold stages and anticontaminator. Our research programs in structural biology involve computerized image analysis of electron micrographs, coordinated with X-ray crystallography, biochemical and biophysical techniques. To make full use of these capabilities, it is essential that we upgrade our electron microscope facilities. At present, there is no microscope on the University of California, San Diego campus that is dedicated to cryo-microscopy. The only microscope on the campus that has a cold stage and anticontaminator is the IVEM JEOL 4000. This microscope is dedicated to electron tomography and is overloaded with users. In contrast, our JEOL 2000 electron microscope experiences less use and this microscope would be extremely suitable for cryo-imaging. The eight major programs requiring low temperature data are: (1) Molecular Structure of Gap Junction Membrane Channels (Sosinsky); (2) Cryomicroscopy of PKA-DAKAP Complexes (Taylor); (3) Cryomicroscopy of PKA-Decorated Cowpea Mosaic Virus (Johnson); (4) Structural Studies of Cellular Components in the Node of Ranvier (Ellisman); (5) Structural Investigation of Bacterial Pathogen Proteins (P. Ghosh); (6) Structural Study of an Enhanceosome (G.Ghosh); (7) Visualization of Recombination Intermediates by Cryo-EM (Segall); (8) Structural Investigations of Conformational Variations Due to Cation or Ligand Binding in Integrins (Stuiver). These eight research projects involve 23 investigators that would depend heavily on the use of a cryo-microscope. In each of these research projects, the primary goal is to determine the three-dimensional structure of organized macromolecular systems and to understand their functions in terms of the dynamic state of their structures. A low-temperature stage would allow samples to be examined in an unstained frozen-hydrated state or if stained, would take advantage of the increased radiation resistance due to the liquid nitrogen temperature. A fully equipped JEOL 2000 would have the full range of these capabilities required for progress in our research programs in structural biology.
