This project involves the acquisition and installation of a 300 kEV field emission gun (FEG) transmission electron microscope (TEM) to determine the structures of cellular machinery. Cellular machines such as the actin cytoskeleton are often large structures difficult to study by methods other than electron cryomicroscopy. For example, it has been possible to dock atomic models for the cytoskeletal components obtained by x-ray crystallography into the molecular maps of the cytoskeletal complexes obtained by electron cryomicroscope. Analysis of filamentous structures has been extended to 10 A resolution in many cases. A state of the art microscope is essential for these studies. The microscope to be obtained and installed during this project will be utilized for the study of the splicesome, voltage-gated ion channels, the actin bundle of the intestinal microvillus, the bacterial flagellar motor and filament, the bacterial gas vesicle, the receptor-kinase signaling complex and the complex of actin with myosin. The aim of these studies are atomic models of the structures. In some instances the structure will be obtained to atomic resolution using electron cryomicroscopy alone. In other cases, atomic models obtained by x-ray crystallography will be docked into molecular maps obtained by cryomicroscopy. The 300 kEV FEG TEM is essential to these projects because the quality of the micrographs limits the resolution of the map in the same way that the quality of the objective lens limits the resolution of the light microscope. No matter how sophisticated, computer processing cannot extract what is not present in the images. The two key features of this microscope in this regard are the higher voltage and the FEG. With these, it is possible to take highly defocused images which have strong contrast at low resolution but which preserve high resolution detail. The strong contrast at low resolution is essential for accurate alignment of images prior to averaging. If the alignment (or correction for distortion) is not accurate, one will average non-equivalent features and resolution will be lost. This microscope will extend the resolution of current maps and therefore, the ability to obtain accurate atomic models whether it be directly or by the docking of atomic models into molecular maps.
The cryomicroscope is essential to the training of young scientists. Since the kind of microscopy being used represents a frontier in structural studies of cellular machinery, students and post doctoral fellows must be trained in these techniques. The need for such scientists is increasing rapidly now that the power of cryoelectron microscopy has been demonstrated. There are currently few places equipped for this kind of training.
