Molecular Structure of SPB Core Proteins

该项目的总体目标是建立必要的实验约束，以构建SPB核心的分子模型，该模型从单个组件的原子结构延伸到它们彼此之间的相互作用以及它们在MTOC中的整体位置，其中信息将由计算核心组装成一个内聚结构。这项结构研究补充了项目2(AGARD)将执行的构成内部和外部斑块的Y-微管蛋白复合体的结构。该试验计划包括三种互补的方法。 第一个目标(Rayment)从SPB各个组成部分的高分辨率X射线结构测定开始，从构成嵌入核膜的中央斑块(Spc110-C、钙调蛋白、Spc29和Spc42-N)的蛋白质和结构域的结构开始，通过中间层2和1(Spc42-C、Cnm67、Nud1和Spc72-C)的结构测定。中间层1形成了连接到外层斑块中的γ-微管蛋白(Tub4)复合体的桥梁，因此这个项目与项目2中描述的γ-微管蛋白的研究相结合并补充了这一研究。目前，SPB核心中的所有蛋白质都已经以适合结构或生物物理研究的可溶形式表达，并且超过一半的蛋白质已经结晶。第二个目标是为天然SPB建立一个分子包膜，高分辨结构可以对接或建模(Agard)。这将通过冷冻电子断层扫描(CryoET)和整个分离SPB的亚体积平均来确定。这将在大约20Å的中间分辨率下提供整个SPB的无偏3D框架。它将揭示每一层中的域组织以及层之间主要SPB组件的交互。蛋白质标签将被用来在图谱中定位和定位单个蛋白质。第三个目标(戴维斯)是为了在各个部件之间产生一组新的距离约束，这些约束是组合来自先前特定目标的信息所需的。将使用两种方法。首先，先前的FRET分析将扩展到中央斑块中的蛋白质，该分析建立了核心中SPB成分的当前排列。其次，通过对原生SPB组件的交联性分析，将建立一组新的高分辨率距离。 这些结构调查将提供各个组成部分的详细结构以及它们在战略规划和预算局中的安排方式。因此，它将建立计算核心为SPB生成伪原子模型所需的信息。反过来，这个三维模型产生的假设将通过项目3和4(Davis，Winey和Rayment)中的生化、遗传和细胞生物学研究进行测试。它还将允许用分子术语解释项目5(Asbury)中测量的微管-SPB附着体的机械强度。

The overall goal of this project is to establish the experimental restraints necessary to construct a molecular model for SPB core that extends from atomic structures of the individual components to their interactions with each other and their overall position in the MTOC where the information will be assembled into a cohesive structure by the computational core. This structural study complements the investigation of the structure of the y-tubulin complexes that constitute the inner and outer plaques that will be performed by Project 2 (Agard). The experimental plan includes three complementary approaches. The first aim (Rayment) begins with high resolution X-ray structural determinations of the individual components of the SPB and proceeds in a logical progression from the structures of the proteins and domains that constitute the central plaque which is embedded in the nuclear envelope (Spc110-C, calmodulin, Spc29, and Spc42-N) through a structural determination of the Intermediate layers 2 and 1 (Spc42-C, Cnm67, Nud1, and Spc72-C). Intermediate Layer 1 forms the bridge to the y-tubulin (Tub4) complex in the outer plaque so that this project interfaces and complements the study of the y-tubulin described in Project 2. At this time all of the proteins in the SPB core have been expressed in a soluble form suitable for structural or biophysical study and more than half of them have been crystallized. The second aim is directed towards establishing a molecular envelope for the native SPB into which the high-resolution structures can be docked or modeled (Agard). This will be established through cryo electron tomography (cryoET) and subvolume averaging of entire isolated SPB. This will provide an unbiased 3D framework of the entire SPB at an intermediate resolution of about 20 Å. It will reveal the domain organization within each layer and the interaction of major SPB components between layers. Protein-tagging will be used to locate and orient individual proteins within the maps. The third aim (Davis) is directed towards generating a new set of distance restraints between individual components that are needed to combine the information from the previous specific aims. Two approaches will be used. First, the prior FRET analysis that established the current arrangement of SPB components in the core will be extended for proteins in the central plaque. Second, a new set of high-resolution distances will be established through crosslinking analysis of native SPB assemblies. These structural investigations will provide detailed structures of the individual components and the manner in which they are arranged in the SPB. Thus, it will establish air of the information necessary for the computational core to generate a pseudo atomic model for the SPB. In turn, hypotheses that arise from this three-dimensional model will be tested through biochemical, genetic and cell biological studies in Projects 3 and 4 (Davis, Winey and Rayment). It will also allow the mechanical strengths of the microtubule-SPB attachments measured in Project 5 (Asbury) to be interpreted in molecular terms.