Overview and Rationale
Structural electron microscopy has become a significant tool in Structural Biology. In this technique, a biological specimen is applied to an EM grid, blotted to remove excess buffer and rapidly plunged in cryogen to embed the particles in a thin film of vitreous buffer. The specimen is maintained at liquid nitrogen temperature when it is transferred into the EM. This provides a frozen-hydrated environment for the specimen in the EM vacuum that preserves the specimen to atomic resolution. This in turn allows high resolution images and diffraction patterns to be acquired at cryogenic temperatures. When combined with image processing, this technique provides 3D electron density maps of complexes, molecular machines and 2D crystals. The resolution of the 3D map obtained with this approach is dependent on a number of factors and can routinely be in the 12-8Å range for single complexes and even higher for 2D crystals. A molecular docking approach is used to place models of individual subunits obtained by X-ray crystallography or NMR within the map, to create a hybrid model that provides insight into function.
In our group, we also determine crystal structures of the components of these machines, using X-ray crystallography (in collaboration with Dr. James Head) and are beginning to use NMR to study small protein domains which cannot be crystallized. There are five major projects in our group that involve studies of large, macromolecular machines.