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

该项目的目的是开发使用电子结晶学分析蛋白质三维结构所需的技术。至于硬波，就是构建一个针对电子结晶学进行优化的电子显微镜。传统的电子显微镜主要是为材料科学家和组织学家设计的。蛋白质晶体的特殊之处在于它们对辐射损伤非常敏感。因此，样品必须保存在冰冻水合的条件下，数据采集应尽可能避免不必要的电子辐射。使用显微镜作为衍射计，可以完成高分辨率的数据采集。为了有效地获取相位数据，必须从相同的场中获取图像。因此，需要将衍射和成像相结合。对现有显微镜进行了许多改进，特别是为了方便和准确地进行低剂量显微镜。为此，我们优化了显微镜固件，并开发了一种新的多点消隐和聚焦方法。电子衍射现在被整合到低剂量过程中。对低温转移和低温检测的几个方面进行了深入的研究。因此，采用了新的泵送系统气体，这将大大减少低温下对样品的冰污染。在软件方面，开发了一套用于确定三维晶体对电子束取向的计算机程序。该程序已应用于肌浆网钙ATPase微晶的研究。

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.