Electron Dense Labels for Macromolecular Imaging

描述（由申请人提供）：蛋白质和多蛋白质组装体的三维（3D）结构的知识对于理解基本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，不能结晶，太大的NMR分析，电子显微镜（EM）是唯一的技术，可以提供有用的结构信息。电子致密分子的附着，包含具有高效率散射电子的重金属元素簇，通常是EM数据收集和分析所必需的。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组有限的附着方法。在许多情况下，这些资源失败了，目标必须被放弃或费力地重新设计。我们建议创造新的电子致密化合物，并开发快速确定将它们连接到靶蛋白质的最佳方法的方法。我们的目标是为EM社区提供一套扩展的蛋白质标记材料和技术，以便更大比例的目标可以在结构上进行表征。这项工作将包括等量的化学合成，化学分析以测量附着反应的整体效率，以及在多功能病毒颗粒平台上进行严格的EM测试。成功的方法将被应用到重要的蛋白质系统，迄今为止，抵制结构表征的电子显微镜。

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.