This proposal is for funds to purchase a new cryo-electron microscope equipped with a slow-scan CCD camera. This system will allow direct acquisition of digital images suitable for computer image reconstruction and structural analysis of macromolecular complexes. Although the UCLA campus has several electron microscopes, most are more than seven years old and none are suitable for state-of-the-art cryo techniques. Each of the major users will contribute different areas of expertise to the shared facility, including cryo sample preparation, classical electron microscopy, site-specific antibody and gold labeling techniques, two- dimensional crystal growth, and three-dimensional image reconstruction. Proposed research projects involve a diverse array of biological assemblies: human adenovirus particles with bound cellular receptors; retroviral capsid protein assemblies; the spliceosome, a complex particle involved in mRNA processing; the vault organelle, a ubiquitous cytosolic ribonucleoprotein particle; cell-to-cell channels, which directly connect the cytoplasms of adjacent cells; and GABAA receptors. The proposed facility will complement the existing strengths at UCLA in imaging and biological structure. The Nuclear Medicine Division has leaders in the fields of PET scanning and medical image reconstruction. The Molecular Biology Institute has several renown x-ray crystallographers. One goal of the proposed facility is to bridge the resolution gap between x-ray crystallography and electron microscopy by combining atomic structural information of component molecules with image reconstructions of macromolecular complexes. The Department of Molecular and Medical Pharmacology will provide suitable renovated space for the cryo-electron microscope. Sufficient computational power for image processing already exists at UCLA. Additional computer graphics workstations will be purchased from the Principal Investigator's start-up funds. After the initial one year warranty period, the ongoing maintenance and operating expenses for the cryo-electron microscope and CCD cameras will be covered jointly by the major users' grants and departmental funds. The administration of the proposed cryo-electron microscopy facility will be carried out by an inter-departmental committee at UCLA. Dr. Nigel Unwin, a recognized leader in the field, has agreed to serve as an external consultant and provide both technical advice and overall scientific guidance.
