Development of Crystal Structure Analysis Methods for Small Protein Crystals Using Anomalous Scattering.

利用反常散射开发小蛋白质晶体的晶体结构分析方法。

基本信息

批准号：
02558011
负责人：
SATOW Yoshinori
金额：
$ 11.2万
依托单位：
UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

The research objective is to develop an X-ray area detector system, utilizing Imaging Plate storage phosphor, for structural studies of small crystals of protein. This study aimed at development of structure determination theory and data processing methods by use of anomalous scattering. Computer programs developed here, as well as other existing structure refinements and graphics programs, were built into a complete system for anomalous diffraction studies.A drum-scanner type X-ray detector was designed and built first. The detector implemented with highly efficient optical system of new design was combined with an Enraf-Nonius oscillation camera for a rotating anode X-ray generator. The detector can be used in oscillation data collection when it is mounted on the horizontal-type four-circle diffractometer system installed at beamline 14A of the Photo Factory synchrotron radiation source at National Laboratory for High Energy Physics. A preliminary application of the detector and program system to a Laue diffraction study was perfomed on the hen egg white lysozyme in order to evaluate feasibility of white radiation anomalous diffraction.Three protein structures of very small crystals were determined based on the synchrotron data measured at several wavelengths. The structure of an Fv fragment from mouse anti-dansyllysine immunoglobulin IgG2a has been determined, finally at 1.6 A^^. resolution. The structure clearly elucidates its complementarity determining regions. Refinement of its crystalline complex with dasyllysine are also in progress. Furthermore, crystal structures of purine-specific ribonuclease U_2 at 1.8 A^^. resolution and of an Fab fragment of 3B44 anti-nitrophenol antibody of IgG12 at 3 A^^. have been determined. These have proved that the developed detector and the program system are widely applicable to structure determination of protein crystals as well as 50 mum.

研究目标是开发一种 X 射线区域探测器系统，利用成像板存储荧光粉，用于蛋白质小晶体的结构研究。本研究旨在发展利用反常散射的结构测定理论和数据处理方法。这里开发的计算机程序以及其他现有的结构改进和图形程序被构建成一个用于异常衍射研究的完整系统。首先设计并建造了鼓式扫描仪型X射线探测器。该探测器采用新设计的高效光学系统，与用于旋转阳极 X 射线发生器的 Enraf-Nonius 振荡相机相结合。该探测器安装在高能物理国家实验室光厂同步辐射源14A束线处的卧式四环衍射仪系统上，可用于振荡数据采集。为了评估白色辐射反常衍射的可行性，对鸡蛋清溶菌酶进行了劳厄衍射研究中的探测器和程序系统的初步应用。根据同步加速器在多个温度下测量的数据，确定了三种非常小的晶体的蛋白质结构。波长。来自小鼠抗丹磺酰赖氨酸免疫球蛋白 IgG2a 的 Fv 片段的结构已确定，最终为 1.6 A^^。解决。该结构清楚地阐明了其互补性决定区域。其与 dasyllysine 的结晶复合物的精制也在进行中。此外，嘌呤特异性核糖核酸酶 U_2 在 1.8 A^^ 处的晶体结构。 IgG12 的 3B44 抗硝基苯酚抗体在 3A^^ 处的 Fab 片段的分辨率。已确定。证明所开发的检测器和程序系统广泛适用于蛋白质晶体以及50μm的结构测定。