Project #1: Harrison

项目摘要(参见说明书)：该项目的这一部分是关于研究三种复杂的分子机器的结构和组装的建议--无包膜病毒、网状蛋白外壳和动粒。(1)我们在无包膜病毒和网状蛋白涂层方面的工作的一个长期目标是将高分辨率结构分析(通过X射线结晶学和单粒子低温电子显微镜)与通过荧光成像对单个粒子的动态进行观察相结合。与计划项目组成员合作确定的近原子分辨率的轮状病毒颗粒和亚纳米分辨率的网状蛋白涂层的低温EM结构，导致了在体外和活细胞中确定病毒进入和网状蛋白组装和剥离机制的实验。我们现在将与项目4合作，使用低温电子显微镜在高分辨率结构分析和活细胞成像之间架起一座桥梁。目标是确定非包膜病毒(这里是轮状病毒)如何穿孔或破坏细胞内小泡的膜，以获得胞浆(这是感染过程中关键且仍然令人费解的一步)，并可视化网状蛋白与其他被膜成分的相互作用。(2)芽期酵母着丝点聚集在着丝粒DNA(-150bp)上，并将每条染色体连接到有丝分裂纺锤体的单个微管(MT)。我们在体外从重组蛋白中重组了DNA近端的“内部动粒”，并测定了它的许多组分的晶体结构。我们将从负染的标本中获得重组的内部动粒及其一些亚复合体的3D重建，并建立这个-750 kDa蛋白质的分子模型：DNA组装。通过对内部动粒结构的分析及其相互作用伙伴的信息，我们将增加连接内部动粒和连接微管的外部动粒成分的连接复合体，并通过EM确定进一步的结构

.PROJECT SUMMARY (See instructions): This component of the Program Project is a proposal to study structure and assembly of three intricate molecular machines ~ a non-enveloped virus, clathrin coats, and kinetochores. (1) A long-term goal of our work on non-enveloped viruses and on clathrin coats has been to connect high-resolution structure analysis (by x-ray crystallography and by single-particle cryoEM) with observations on the dynamics of individual particles by fluorescence imaging. CryoEM structures of rotavirus particles at near-atomic resolution and of clathrin coats at subnanometer resolution, determined in collaboration with members of the Program Project team, have led to experiments that define mechanisms of viral entry and clathrin assembly and uncoating, both in vitro and in living cells. We will now use cryoET to bridge between high-resolution structural analyses and live-cell imaging, in collaboration with Project 4. The objectives are to determine how a non-enveloped virus (rotavirus in the work here) perforates or disrupts the membrane of an intracellular vesicle in order to gain access to the cytosol ~ a critical and still puzzling step in the infectious process ~ and to visualize interactions of clathrin with other coat components. (2) Budding-yeast kinetochores assemble on centromeric DNA (-150 bp) and connect each chromosome to a single microtubule (MT) of the mitotic spindle. We have reconstituted the DNA-proximal "inner kinetochore" from recombinant proteins in vitro, and we have determined crystal structures of many of its components. We will obtain 3D reconstructions, from negatively stained preparations and by cryoEM where appropriate, of the reconstituted inner kinetochore and some of its subcomplexes, and build a molecular model of this -750 kDa protein:DNA assembly. From analysis of the inner-kinetochore structure and information on its interaction partners, we will add additional "linker" complexes, which connect the inner kinetochore with microtubule-attached "outer kinetochore" components, and determine further structures by EM