Improvement of High resolution data collection in macromolecular electron cryo-microscopy

电子冷冻显微镜和图像分析已经成为 用于确定三维结构的有用工具 在近原子分辨率的大分子组装。 我们 实验室配备了400千伏电子 由国家研究中心资助的显微镜 国家卫生研究院作为国家机构的资源。 以补充我们 在NIH的支持下，这项提案旨在改善 标本制备程序，冷冻标本保持器 我们的400 kV电子低温系统的性能和数据收集， 显微镜 这项技术的前半部分 开发将与Gatan合作进行 Inc.建立一个碳低温镀膜机真空存款， 薄碳膜上冻结，水合标本，和高 JEOL 400 kV电子用高分辨率低温试样保持器 显微镜 该设备的性能将是 使用各种生物样本进行评估， 在我们的资源中心进行调查。 的最终版本 该设备将成为嘉坦的标准产品。 的 该提案的第二部分是开发软件， 捕获和分析电子衍射图案， 生物标本的图像与我们新获得的 1024x1024慢扫描电荷耦合器件（CCD）相机。 该软件将用于研究 社区 这些先进的技术将用于 我们国家卫生研究院的几个合作和用户项目支持 资源中心。

Electron cryo-microscopy and image analysis have become useful tools for determining 3-dimensional structures of macromolecular assemblies at near atomic resolution. Our laboratory is uniquely equipped with a 400 kV electron microscope funded by the National Center for Research Resources of NIH as a national facility. To complement our NIH support, this proposal is aimed at improving the specimen preparation procedure,, cryo-specimen holder performance and data collection on our 400 kV electron cryo- microscope. The first half of this proposed technology development will be carried out in collaboration with Gatan Inc. to build a carbon cryo-coater for vacuum deposit of a thin carbon film onto frozen, hydrated specimens, and high resolution cryo-specimen holder for the JEOL 400 kV electron microscope. The performance of this equipment will be evaluated with a variety of biological specimens under investigation in our Resources Center. The final version of this equipment will become standard products of Gatan. The second half of this proposal is to develop software for capture and analysis of electron diffraction patterns and images of biological specimens with our newly acquired 1024x1024 slow scan charge-coupled device (CCD) camera. This software will be made available to the research community. These enchanced technologies will be used for several collaborative and user projects in our NIH supported Resource Center.