3-D ELECTRON MICROSCOPY OF MACROMOLECULES

Our objectives are to determine the three-dimensional (3D) crystal
structures of macromolecules at near atomic resolution and the 3-D
structures of non-crystalline macromolecular assemblies at intermediate
resolution, by a combination of electron microscopy and computer processing
methods. The present proposal is a resource related project. Requests
include the acquisition of a medium high voltage (300 kV) electron
microscope with appropriate accessories and an operational fund to develop
the technology for the above aims. Technical development will be focused
on specimen preparation, 3-D data collection at low dose and low
temperature conditions and implementation of computer programs for 3-D
reconstruction. The developed technology will be immediately applied to
the high resolution crystal structure determination of crotoxin complex, T4
DNA helix destabilizing protein, fungus lysozyme and Fc fragments; and to
the intermediate resolution structure determination of muscle Z disc and
flagella axoneme. Collaborative projects will be established with
investigators outside our research groups after the midpoint of the grant
period. We plan to complete a thorough analysis of the potential as well
as limitations of this technology in its general applicability to a variety
of biological systems. We expect to present a final recommendation on the
feasibility and desirability of establishing a national shared facility of
this technology at the end of this grant period.