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，其中信息将通过计算核心组装成内聚结构。该结构研究补充了将由项目2（Agard）进行的构成内外斑块的γ-微管蛋白复合物结构的研究。该实验计划包括三个互补的方法。 第一个目标（Rayment）始于SPB各个组分的高分辨率X射线结构测定，并从构成包埋在核被膜中的中央斑块的蛋白质和结构域的结构开始进行逻辑进展通过中间层2和1（Spc42-C、Cnm67、Nud1和Spc72-C）的结构测定，对中间层2和1（Spc110-C、钙调蛋白、Spc29和Spc42-N）进行了结构分析。中间层1形成了与外斑块中的γ-微管蛋白（Tub4）复合物的桥梁，因此该项目与项目2中描述的γ-微管蛋白研究相互作用并相互补充。此时，SPB核心中的所有蛋白质都以适合于结构或生物物理研究的可溶形式表达，并且其中一半以上已经结晶。第二个目标是为天然SPB建立分子包膜，高分辨率结构可以对接或建模到其中（Agard）。这将通过冷冻电子断层扫描（cryoET）和整个孤立SPB的子体积平均值来确定。这将以大约20 μ m的中间分辨率提供整个SPB的无偏3D框架。它将揭示每个层中的域组织以及层之间主要SPB组件的交互。蛋白质标记将用于定位和定向地图内的个别蛋白质。第三个目标（Davis）是针对在各个组件之间生成一组新的距离约束，所述距离约束是联合收割机组合来自先前特定目标的信息所需的。将采用两种方法。首先，先前的FRET分析建立了核心中SPB组分的当前排列，将扩展到中央斑块中的蛋白质。第二，一组新的高分辨率的距离将建立通过交联分析本地SPB组件。 这些结构调查将提供单个组件的详细结构以及它们在SPB中的布置方式。因此，它将建立计算核心生成SPB的伪原子模型所需的信息。反过来，从这个三维模型中产生的假设将通过项目3和4（Davis，Winey和Rayment）的生物化学，遗传学和细胞生物学研究进行测试。它还将允许在项目5（阿斯伯里）中测量的微管-SPB连接的机械强度以分子术语解释。

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.