High throughput icosahedral particle reconstruction

描述（由申请人提供）：我们的实验室通过结合电子低温显微镜记录的数千个粒子图像，展示了解决几个二十面体粒子中分辨率（7-9埃）结构的能力。在这些分辨率下，我们已经能够识别出蛋白质的大部分螺旋和薄片。目前，即使对于经验丰富的用户，数据收集和计算机图像重建的过程也是非常劳动密集型的，对于新用户来说，学习曲线非常陡峭。我们建议开发一种高通量程序，以便在3-5周内完成中分辨率二十面体粒子的结构测定过程-从数据收集到结构测定和解释。这意味着完成每个项目的时间减少了10-20倍。这一技术进步将允许分子病毒学家使用这种结构作为标准方法来理解病毒组装背景下的结构和功能关系以及与细胞因子（如抗体和受体）的相互作用。这项建议包括两个发展部分。首先，在新获得的200千伏电子低温显微镜上，利用4k × 4k CCD相机进行了半自动在线数字图像采集。二是提高现有软件的计算效率，整合许多独立软件，包括颗粒拾取、显微镜参数确定、颗粒取向和中心细化、三维重建和结构解释。我们将使用Python脚本语言来协调这些程序，并使用面向对象的数据库来管理整个项目数据采集和分析过程中的元数据。该软件将免费提供给研究界。我们将把这些技术改进部署到我们已经资助的P22噬菌体和疱疹病毒项目，以及委内瑞拉马脑炎病毒（一种具有潜在生物恐怖主义威胁的选定制剂）的新项目中。

DESCRIPTION (provided by applicant): Our laboratory has demonstrated the capability of solving medium resolution (7-9 Angstroms) structures of several icosahedral particles by combining several thousand-particle images recorded in an electron cryomicroscope. At these resolutions, we have been able to identify most of the alpha helices and beta sheets of the proteins. Presently, the process of data collection and computer image reconstruction is very labor intensive, even for experienced users, and has a very steep learning curve for new users. We propose to develop a high throughput procedure so that the process of structure determination of icosahedral particles at medium resolution - from data collection to structure determination and interpretation - can be completed within 3-5 weeks. This represents a factor of 10-20x reduction in time for completion of each project. This technological advance will allow molecular virologists to use such structures as a standard approach to understand structure and function relationship in the context of virus assembly and interactions with cellular factors such as antibodies and receptors. This proposal consists of two developmental components. The first is to develop semi-automatic on-line digital image acquisition with a 4k x 4k CCD camera in our newly acquired 200 kV electron cryomicroscope with a field emission gun. The second is to improve the computational efficiency of existing software and to integrate much of the stand-alone software, which includes particle picking, microscope parameter determination, particle orientation and center refinement, 3D reconstruction and structural interpretation. We will use the Python scripting language to coordinate these programs and use an object-oriented database to manage the metadata throughout the project data acquisition and analysis. This software will be made freely available to the research community. We will deploy these technology enhancements to our already funded projects on P22 bacteriophage and herpes virus in addition to a new project with the Venezuelan Equine Encephalitis virus, a select agent with potential bioterrorism threat.