Project #1: Harrison

项目

• 批准号: 8462406
• 负责人: STEPHEN COPLAN HARRISON
• 金额: $ 75.9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462406
• 关键词: Architecture; Binding; Biological; C-Microtubule; Capsid; Cell division; Cells; Chromosome Segregation; Chromosomes; Clathrin; Collaborations; Complex; Computing Methodologies; Cryoelectron Microscopy; Crystallography; Cytosol; DNA; Electron Microscopy; Event; Goals; Image; Imaging technology; In Vitro; Individual; Instruction; Intracellular Membranes; Investigation; Kinetochores; Life; Link; Maps; Mediating; Membrane; Methods; Microtubules; Mitotic spindle; Modification; Molecular; Molecular Machines; Molecular Models; Pathway interactions; Penetration; Preparation; Procedures; Process; Proteins; Recombinant Proteins; Recombinants; Recruitment Activity; Resolution; Rotavirus; Saccharomycetales; Staining method; Stains; Structure; Surface; System; Testing; Toxin; Vesicle; Viral; Virion; Virus; Work; Yeasts; base; cellular imaging; conformational conversion; density; fluorescence imaging; image reconstruction; insight; member; molecular modeling; particle; pathogen; programs; reconstitution; reconstruction; research study; uptake

.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

项目概要（见说明）：该项目的这一部分是一项研究三种复杂分子机器的结构和组装的建议-无包膜病毒，网格蛋白外套和动粒。(1)我们对无包膜病毒和网格蛋白外壳的工作的长期目标是将高分辨率结构分析（通过X射线晶体学和单粒子cryoEM）与通过荧光成像对单个粒子的动态观察相连接。冷冻电镜结构的轮状病毒颗粒在近原子分辨率和网格蛋白涂层在亚纳米分辨率，确定在合作与程序项目组的成员，导致实验，定义机制的病毒进入和网格蛋白组装和脱壳，在体外和活细胞。我们现在将与项目4合作，使用cryoET在高分辨率结构分析和活细胞成像之间架起桥梁。目的是确定无包膜病毒（本文中的轮状病毒）如何穿透或破坏胞内囊泡的膜，以进入胞质溶胶-感染过程中的关键且仍然令人困惑的步骤-并可视化网格蛋白与其他外壳组分的相互作用。(2)芽殖酵母动粒在着丝粒DNA（~ 150 bp）上组装，并将每条染色体连接到有丝分裂纺锤体的单个微管（MT）。我们已经重建了DNA近端的“内动粒”，从体外重组蛋白质，我们已经确定了它的许多组件的晶体结构。我们将获得3D重建，从负染色的制剂和cryoEM在适当的情况下，重建的内部动粒和它的一些亚复合物，并建立一个分子模型，这个约750 kDa的蛋白质：DNA组装。通过分析内部动粒结构及其相互作用伙伴的信息，我们将添加额外的“连接体”复合物，将内部动粒与微管连接的“外部动粒”组分连接起来，并通过EM确定进一步的结构