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

项目摘要 TR&D项目3.分析阶段II：用于分析连接性和形态的工具 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复合物。为此，我们的分子显微镜管道需要 关于装配体零部件的形状和连接性的信息，准确地表示它们的 以最高的空间和时间分辨率排列。因此，我们将开发和完善方法， 确定复合物内组分在几个距离处的形态和空间关系 尺度，范围从总体子单元形状和排列的描述到定义原子分辨率 大分子对之间的接触。我们的策略需要使用正交方法，以提供 补充数据，并涵盖了广泛的分辨率，告诉我们的形状，尺寸和 单个蛋白质和大分子组装体的连接性。我们将重点关注已经 事实证明，这些建议特别具有授权作用，但仍有很大的进一步发展空间。这些包括 电子显微镜（EM）和化学交联质谱（XL-MS）：通过前者， 可以产生具有足够细节的形态图以解析复合体的形状和位置， 蛋白质，结构域和折叠;平行地，通过后者，我们将获得关于蛋白质，结构域和折叠的每个组成部分的信息。 该组件相对于所有其它部件和整个结构定位。这些数据结合起来 与数据互补，完善的方法，将用于生成结构模型， 组件.为了阐明细胞功能，我们建议收集和解释有关细胞功能变化的动态数据， 装配体的形态，以及这些装配体内不断变化的相互作用。分子显微镜 因此，pipeline寻求构建具体的高精度3D模型和随时间变化的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.