Midwest Consortium for High Resolution Cryoelectron Microscopy

项目总结 近年来，结合三维重建的单粒子冷冻电子显微镜(Cryo-EM)技术应运而生 作为一种革命性的工具，用于解决病毒和大分子复合体的高分辨率三维结构。 使用低温EM解决的近原子分辨率(3-4Ǻ)结构的数量迅速增加，使得 前所未有的对基本细胞过程和病毒感染的原子水平的了解。为了获得 近原子分辨率低温电磁结构，需要使用状态收集高分辨率图像数据 最先进的成像资源，包括高端透射式电子显微镜和直接电子显微镜 探测器。直接电子探测器不仅提高了图像的分辨率和对比度，而且还可以记录电影 以便随后对电子束引起的样品在曝光期间的移动进行计算校正。这个 提高图像的对比度和分辨率是解决小分子的近原子分辨率结构的关键 蛋白质复合体。然而，购买和维护最先进的低温EM资源的高昂成本， 无论是高端电子显微镜还是直接电子探测器，都排除了许多低温电磁研究人员 无法接触到这些新技术。在这里，创建一个中西部高中联盟- 分辨率低温电子显微镜的提出提供了获取这种高分辨率数据的途径 在没有获得这种资源的情况下，为低温电磁实验室提供能力。该财团将由4名成员组成 来自主办机构(普渡大学)的调查人员它将保持高分辨率的数据收集 资源(Titan Krios 300KVFEG显微镜，带相位板、能量过滤器和直接电子探测器)和 将向来自10家合作机构(波士顿大学)的11名调查人员提供服务医学院， 密歇根州立大学，宾夕法尼亚州立医学院，罗格斯大学。阿拉巴马州大学伯明翰分校 科罗拉多州博尔德大学来自堪萨斯大学。密苏里州州立大学佛蒙特州大学和弗吉尼亚联邦大学)。 普渡工厂员工、教职员工和现场服务工程师的高分辨率集体体验 低温电磁成像，将确保设施以最高性能运行，并将服务中断降至最低。 普渡大学建立的、合作实验室可以访问的高分辨率数据收集能力，将 允许这些调查人员，他们都是，除了3名新的调查人员，由NIH资助，以克服 分辨率障碍并促进在他们自己的低温电磁项目中对一系列结构的发现，例如， 细菌病原体黏附蛋白，人类病毒，亨廷顿病蛋白，突触小泡蛋白， 化学感受器信号复合体等。联合体将为低温EM提供全面支持 项目，包括运送样品，准备样品网格，收集高分辨率单粒子和 断层扫描图像，处理原始电影，并将数据传输回合作伙伴的实验室。数据 收集服务的范围从完整的数据收集，合作伙伴只需邮寄样本，然后 接收图像数据，并由由托管设施工作人员培训的合作伙伴进行冷冻EM的实际操作。

PROJECT SUMMARY Recently, single particle cryo-electron microscopy (cryo-EM) combined with 3-D reconstruction has emerged as a revolutionary tool for solving high-resolution 3-D structures of viruses and macromolecular complexes. The rapidly increasing number of near-atomic resolution (3-4Ǻ) structures solved using cryo-EM has allowed unprecedented atomic level understanding of fundamental cellular processes and viral infections. To obtain near-atomic resolution cryo-EM structures, requires the collection of high-resolution image data using state-of- the-art imaging resources, including both a high-end transmission electron microscope and a direct electron detector. The direct electron detectors not only improve image resolution and contrast, but also record movies for subsequent computational correction of electron beam-induced, sample movements during exposure. The increased image contrast and resolution are essential for solving near-atomic resolution structures of small protein complexes. However, the high cost to purchase and maintain a state-of-the-art cryo-EM resource, with both a high-end electron microscope and a direct electron detector, precludes many cryo-EM investigators from having access to these new techniques. Here, the creation of a Midwest Consortium for High- Resolution Cryo-electron Microscopy is proposed to provide access to such high-resolution data collection capability for cryo-EM laboratories without access to such resources. This consortium will consist of 4 investigators from the host institution (Purdue Univ.) which will maintain the high-resolution data collection resource (Titan Krios 300kV FEG microscope with phase plate, energy filter, and direct electron detector) and will provide services to 11 investigators from 10 partnering institutions (Boston Univ. School of Medicine, Michigan State Univ., Penn State College of Medicine, Rutgers Univ., Univ. of Alabama at Birmingham, Univ. of Colorado Boulder, Univ. of Kansas, Univ. of Missouri, Univ. of Vermont, and Virginia Commonwealth Univ.). The collective experience of the Purdue facility staff, faculty and onsite service engineer, in high-resolution cryo-EM imaging, will ensure that the facility operates at peak performance with minimal service interruptions. The high-resolution data collection capabilities established at Purdue and accessible to the partnering labs, will allow these investigators, who are all, except for the 3 new investigators, NIH-funded, to overcome the resolution barrier and facilitate discovery within their own cryo-EM projects on a range of structures, such as, bacterial pathogen adhesion proteins, human viruses, Huntington's Disease proteins, synaptic vesicle proteins, chemoreceptor signaling complex, etc. The Consortium will provide comprehensive support to the cryo-EM projects, including shipping samples, preparing sample grids, collecting high-resolution single particle and tomography images, processing raw movies, and transferring data back to the partners' labs. The data collection services will range from full data collection where partners simply mail in the samples and then receive the image data, to hands-on operations of the cryo-EM by partners trained by the host facility's staff.