Development of technologise for three-dimensional structural analysis of protein crystals using electron microscope

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The aim of this project is to develop technologies necessary for analyzing three-dimensional structure of proteins using electron crystallography. As to the hardwave, it is to construct an electron microscope optimized for electron crystallography. Conventional electron microscopes have been designed for mostly material scientists and histologists. Protein crystals are special in that they are very sensitive to radiation damage. Hence specimens must be kept in frozen-hydrated conditions and data taking should avoid unnecessary electron irradiation as much as possible. High resolution data collection can be done using the microscope as a diffractometer. To obtain phase data efficiently, image must be obtained from the same field. Thus integration of diffraction and imaging is needed. Many modifications to the existing microscope were made, in particular to allow easy and accurate low dose microscopy. We have optimizad microscope firmware to do so and developed a new method for blanking and focusing at multiple points. Electron diffraction is now integrated in the low-dose procedure. Several aspects of the cryo-transfer and low temperature examination were examined intensively. As a result, new pumping system gas been employed, which will greatly reduce ice contamination to the specimen at low temperature. As to the software side, a set of computer program has been developed for determining the orientation of a three-dimensional crystal to the electron beam. The program has been applied to micro crystals of calcium ATPase of sarcoplasmic reticulum.