Project #1: Harrison

．项目概述（见说明）：本项目的这一部分是研究三种复杂分子机器的结构和组装的建议：非包膜病毒、网格蛋白外壳和着丝点。我们研究非包膜病毒和网格蛋白外壳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低温电子显微镜）与荧光成像对单个粒子动力学的观察联系起来。轮状病毒颗粒在近原子分辨率下的低温电镜结构和网格蛋白外壳在亚纳米分辨率下的低温电镜结构是与项目团队成员合作确定的，这导致了在体外和活细胞中定义病毒进入和网格蛋白组装和脱壳机制的实验。现在，我们将与Project 4合作，使用cryoET在高分辨率结构分析和活细胞成像之间建立桥梁。目的是确定非包膜病毒（这里的工作是轮状病毒）如何穿透或破坏细胞内囊泡的膜以进入细胞质（这是感染过程中一个关键但仍令人困惑的步骤），并可视化网格蛋白与其他外壳成分的相互作用。(2)发酵菌着丝点聚集在着丝粒DNA上（- 150bp），并将每条染色体连接到有丝分裂纺锤体的单个微管（MT）上。我们在体外用重组蛋白重建了dna近端“内着丝点”，并确定了其许多组分的晶体结构。我们将获得三维重建，从负染色的准备和低温低温下，重建的内部着丝点和它的一些亚复合物，并建立这个-750 kDa蛋白质的分子模型：DNA组装。通过分析内部着丝点的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，将内部着丝点与微管附着的“外部着丝点”组分连接起来，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