Acquisition of 200keV FEG Cryo-Electron Microscope

University Of Virginia Main Campus, Charlottesville VA

Investigators

Abstract

ABSTRACT 0002805 Edward H. Egelman University of Virginia Acquisition of 200 KeV FEG Cryo-Electron Microscope Electron microscopy (EM) is an extremely powerful technique for studying the structure of macromolecular complexes, such as protein polymers, protein-DNA complexes and viruses. Significant advances in electron microscopy have been made in specimen preparation, instrumentation and image. These advances make it possible to use an electron microscopic to determine the three-dimensional structure of a protein. In addition, they also allow for lower-resolution studies that are complementary to those obtained by such techniques as x-ray crystallography and nuclear magnetic resonance. A 200 KeV Field Emission Gun cryo-electron microscope, manufactured by Philips/FEI, will significantly enhance the research programs of a group of six investigators at the University of Virginia Medical School. This microscope will have a highly coherent electron source that will allow for resolutions much higher than obtainable with conventional instruments. The microscope will be equipped for the imaging of frozen-hydrated specimens, maintained in the microscope at liquid nitrogen temperatures. It will also have image acquisition capabilities, using a CCD camera, that will allow for on-line image analysis. 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; 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; 3) studies of the icosahedral Herpes Simplex virus capsid; 4) studies of Ca2+-dependent membrane binding proteins such as annexin, using two dimensional crystals formed on lipid monolayers; 5) studies of Protein Kinase C isozymes using two dimensional crystals formed on lipid monolayers; 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. Many of the projects have already produced results that are at the limit of reliable resolution obtainable by conventional TEM of negatively stained specimens. The new instrument will not only enhance these individual research programs, but will greatly strengthen the environment for structural biology at the University of Virginia and the training of graduate students and postdoctoral fellows.