A 200keV Field Emission Gun (FEG) cryo-electron microscope will significantly enhance the research programs of a group of six investigators at the University of Virginia Medical School. The projects that will take advantage of the increased resolution provided by this instrument are: 1) studies of F-actin, and complexes of F-actin with actin- binding proteins using a single-particle approach to surmount the disorder within these filaments (Egelman); 2) studies of protein-DNA complexes, particularly helical recombination filaments such as RecA/Rad51/UvsX, and ring helicases such as rho, DnaB and T7 gp4 (Egelman); 3) studies of the icosahedral Herpes Simplex virus capsid (Brown); 4) studies of Ca2- dependent membrane binding proteins such as annexin, using two dimensional crystals formed on lipid monolayers (Sando, Kretsinger); and 6) studies of ion channels, such as VacA hexamers, using cryo-EM of two-dimensional crystals to complement ongoing studies with Atomic Force Microscopy. All of the projects require such a microscope with a coherent source to go beyond the resolution that can be obtained in a conventional EM. Many of the projects have already produced results that are at the limit of reliable resolution obtainable by conventional TEM of negatively stained specimens. Imaging processing facilities are now in place at the University of Virginia to fully exploit the data obtained. Further, this microscope will be the center of a facility that is badly needed, since most of the coPI's do not have ready access to another EM. This instrument will not only enhance these individual research programs, but will greatly strengthen the environment for structural biology at the University of Virginia.
