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Development of electron diffraction methods for protein crystallography

蛋白质晶体学电子衍射方法的发展

基本信息

批准号：
10558106
负责人：
TOYOSHIMA Chikashi
金额：
$ 7.3万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Rotation camera and related methods for electron diffraction from thin protein crystals have been developed for facilitating the structure determination of membrane proteins of biological importance. This involved many modifications of the microscope and developments of computer control software and high tilt angle cryospecimen holder. Also developed was a set of programmes for indexing of reflections, integration and scaling of electron diffraction intensities. These developments were necessary because the diffraction patterns from multilayer crystals should be treated as those from 3-dimensional crystals, yet cannot be analysed by conventional X-ray crystallography.First version of the rotation camera used DC motor and integrated rotary encoder, and slow-scan CCD detector. This system was constructed without much investment and can be applied to most of modern electron microscopes. This system was applied to ultrathin crystals of Ca^<2+>-ATPase and yielded preliminary data to ±30゜ of tilt. By using"minimum dose electron diffraction procedures"developed in this study, up to 100 diffraction patters (rotation images) could be recorded from each area. The second version consisted of Heidenhein's high-precision rotary encoder and pulse motor. The hardware Was assembled but the control software was incomplete and yet to be developed. Nevertheless, the preliminary results obtained are very promising.

旋转照相机和相关的方法，从薄的蛋白质晶体的电子衍射已被开发，以促进结构测定的膜蛋白的生物学意义。这涉及到显微镜的许多修改和计算机控制软件和高倾角冷冻标本保持器的发展。还开发了一套用于反射指数化、电子衍射强度积分和缩放的程序。这些发展是必要的，因为多层晶体的衍射图案应被视为那些从三维晶体，但不能通过传统的X射线晶体学进行分析。第一个版本的旋转相机使用直流电机和集成的旋转编码器，和慢扫描CCD检测器。该系统投资少，可应用于大多数现代电子显微镜。将该系统应用于Ca^<2+>-ATP酶晶体的倾斜测量，获得了±30 °倾斜的初步数据。通过使用本研究中开发的“最小剂量电子衍射程序”，可以从每个区域记录多达100个衍射图案（旋转图像）。第二个版本由Heidenhein的高精度旋转编码器和脉冲电机组成。硬件已经组装好，但控制软件还不完整，有待开发。尽管如此，所取得的初步结果是非常有希望的。