Molecular electron cryo-microscopy (cryoEM) is at the same stage of development today as macromolecular x-ray crystallography was in 1980. We are entering an exponential growth phase of this technology accompanied by significant expansion in the number of practitioners and applications. Publications describing the utilization molecular cryoEM in combination with crystallography have grown dramatically and they have revealed exceptionally interesting features of mechanistic biology. Molecular cryoEM fills a unique niche between high resolution cytological EM and crystallography. Examining specimens not immobilized in a crystal lattice has allowed the characterization of dynamic features of macromolecular assemblies impossible to characterize by crystallography and to determine sub nanometer structures of species too large or too heterogeneous to be crystallized. A remarkably powerful strategy has been to determine high-resolution structures of the components of a macromolecular complex by crystallography or NMR followed by the construction of a pseudo atomic resolution model based on the cryoEM reconstruction of the intact organization. At resolutions currently achievable with high voltage FEG instruments, the precision of this modeling into cryoEM density is exceptionally high. This application requests partial support from NIH for a 300KV electron microscope equipped with a field emission gun, a helium cryo stage and a 4k by 4k slow scan CCD. This instrument will fill a gap that exists on the Torrey Pines Mesa in the atoms to cells continuum of structural studies that is the backbone of the world-class biological science produced by the participating institutions; University of California, San Diego; The Burnham Institute and The Scripps Research Institute. Projects proposed for high resolution work with this instrument include molecular motors, membrane proteins and their interactions, complexes controlling intercellular communication and virus assembly and maturation. In addition, software and hardware developed for high throughput EM data collection will be implemented on the requested microscope as it is perfected. These are all well funded programs that have benefited from moderate resolution studies and will immediately utilize the new technology when it is available. We anticipate that a broad range of projects from the Torrey Pines Mesa will find this technology useful as investigators not currently directly involved with molecular cryoEM are introduced to results emerging from its use. ? ? ?
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