TR&D Project 3. The Analysis Stage II: Tools for Analyzing the Connectivity and Morphology of Macromolecular Assemblies

项目总结 R&D项目3.分析阶段II：用于分析连通性和形态的工具 大分子组装体 由于程序集的结构与其功能角色直接相关，因此我们的目标是定义 分离的感兴趣的天然大分子络合物。为此，我们的分子显微镜流水线需要 有关部件零部件的形状和连接性的信息，准确表示其 以最高的空间和时间分辨率进行排列。因此，我们将开发和改进方法，以 确定几个距离处复合体内部组件之间的形态和空间关系 尺度，从描述整个亚基的形状和排列到定义原子分辨率 大分子对之间的接触。我们的战略需要使用正射方法，以便提供 补充数据并涵盖广泛的分辨率，以告知我们有关形状、尺寸和 单个蛋白质和大分子组件的连接性。我们将把重点放在已经 事实证明，这特别有能力，但有很大的进一步发展的余地。这些措施包括 电子显微镜(EM)和化学交联质谱(XL-MS)：通过前者，我们 可以产生足够详细的形态地图来解析复合体的形状和位置， 蛋白质、结构域和折叠；同时，通过后者，我们将获得关于每个成分如何 部件相对于所有其他零部件和整个结构进行定位。将这些数据合并在一起 使用来自补充的、完善的方法的数据，将被用于生成 装配。为了阐明细胞功能，我们建议收集和解释关于变化的动态数据 组件的形态，以及这些组件内不断变化的相互作用。我们的分子显微镜 因此，管道寻求建立具体的高精度3D模型，以及随时间变化的4D模型。

PROJECT SUMMARY TR&D Project 3. The Analysis Stage II: Tools for Analyzing the Connectivity and Morphology of Macromolecular Assemblies As the structure of an assembly is directly related to its functional role, we aim to define the architecture of isolated native macromolecular complexes of interest. For this, our molecular microscope pipeline needs information on the shape and connectivity of an assembly’s components, accurately representing their arrangement at the highest spatial and temporal resolution. Thus, we will develop and refine methodologies to determine the morphologies and spatial relationships between components within complexes at several distance scales, ranging from a description of the overall subunit shape and arrangement to defining atomic resolution contacts between pairs of macromolecules. Our strategy entails using orthologous methods, in order to provide complementary data and to cover a wide range of resolutions, to inform us about the shape, dimensions and connectivity of single proteins and macromolecular assemblies. We will focus on methods that have already proven particularly empowering, but which have significant scope for further advancement. These include electron microscopy (EM) and chemical cross-linking with mass spectrometry (XL-MS): through the former, we can produce morphological maps with sufficient detail to resolve the shapes and locations of complexes, proteins, domains and folds; in parallel, through the latter, we will obtain information on how each component of the assembly is positioned relative to all other components, and the entire structure. These data when combined with data from complementary, well-established methods, will be used to generate structural models of assemblies. To elucidate cellular functions, we propose to gather and interpret dynamic data about the changing morphologies of assemblies, and the changing interactions within these assemblies. Our molecular microscope pipeline thus seeks to build concrete high precision 3D models, and 4D models that change in time.