Electron Dense Labels for Macromolecular Imaging

描述(由申请人提供)：了解蛋白质和多蛋白质组合的三维(3D)结构对于理解基本的生物学过程和设计更有效的药物非常重要。对于许多这样的物种，这些物种无法结晶，而且太大，无法进行核磁共振分析，电子显微镜(EM)是唯一可以提供有用结构信息的技术。电子致密分子的附着通常是电磁数据收集和分析所必需的，因为分子中含有能高效散射电子的重金属元素簇。高分辨率EM的从业者通常只能从商业供应商那里获得两种电子密度材料和一套有限的连接方法。在许多情况下，这些资源会失败，目标要么被放弃，要么被费力地重新设计。我们建议创造新的电子致密化合物，并开发方法来快速确定将它们连接到目标蛋白质的最佳方式。其目标是为EM社区提供一套扩展的蛋白质标记材料和技术，以便能够对更大比例的目标进行结构表征。这项工作将包括等量的化学合成、测量附着反应总体效率的化学分析，以及在多功能病毒颗粒平台上进行严格的EM测试。然后，成功的方法将被应用于重要的蛋白质系统，这些系统到目前为止一直抵制通过电子显微镜进行结构表征。

DESCRIPTION (provided by applicant): Knowledge of the three-dimensional (3D) structures of proteins and multi-protein assemblies is of great importance to the understanding of fundamental biological processes and the design of more effective Pharmaceuticals. For many such species, which cannot be crystallized and are too large for NMR analysis, electron microscopy (EM) is the only technique that can provide useful structural information. The attachment of electron-dense molecules, containing clusters of heavy metallic elements that scatter electrons with high efficiency, is often necessary to EM data collection and analysis. Practitioners of high-resolution EM usually have access to only 2 electron-dense materials and a limited set of attachment methods available from commercial suppliers. In many cases, these resources fail and the target must be either abandoned or laboriously re-engineered. We propose to create new electron-dense compounds and develop methods to rapidly identify the best ways to connect them to target proteins. The goal is to give the EM community an expanded set of materials and techniques for protein labeling, so that a greater fraction of targets can be structurally characterized. The effort will be comprised of equal parts chemical synthesis, chemical analysis to measure the overall efficiency of attachment reactions, and rigorous EM testing on a versatile virus particle platform. Successful methods will then be applied to important protein systems that have thus far resisted structural characterization by electron microscopy.