This application proposes the purchase of Philips CM300-FEG electron microscope with a maximum 300 kev accelerating voltage and field emission illumination. The CM300-FEG is fully computerized with a revolutionary goniometer and capabilities that are particularly well designed for electron tomography and cryoelectron microscopy (cryoEM). A specialized holder and anticontaminator will be purchased to facilitate cryoEM of biological specimens and a CCD camera from Gatan with Macintosh computer system to facilitate development of computerized on-line data collection and to obtain instant feedback on the quality of specimen preparation. The capabilities of this instrument will enhance the research of 6 full time faculty who use electron microscopy as an integral part of their research. Research projects include electron tomography of insect flight muscle (Taylor), electron crystallography of actinin (Taylor) and the Ca2+-ATPase (Taylor), electron diffraction of oriented gels of tobacco mosaic virus (Caspar), 3-D reconstruction of helical filaments of the major sperm protein (MSP) from Ascaris suum (Roberts & Stewart), 3-D reconstruction of filamentous bacteria viruses and pili (Makowski), studies of the 3-D geometry of immune complexes (Roux) and studies on the structure of the cytoskeleton (Keller). This instrument will enhance these projects (1) through high resolution imaging of thicker specimens, (2) through digital survey of specimens prior to data collection by means of on-line image recording, (3) by enabling development of protocols for automated tilt series data collection for tomography by virtue of its motorized and computerized specimen stage (4) by facilitating electron diffraction of oriented, multilayered arrays of filaments, (5) by facilitating high resolution imaging of 2-D crystalline and paracrystalline arrays of proteins and multiprotein complexes, (7) by enabling high resolution imaging of single molecules and small complexes by utilizing the high c oherence of the field emission electron source and the high depth of focus provided by 300 kev electrons.
