Transmission Electron Microscope

描述(申请人提供)：分子电子冷冻显微镜(CryoEM)与1980年的大分子X射线结晶学处于相同的发展阶段。我们正在进入这项技术的指数增长阶段，同时从业人员和应用程序的数量也在显著增加。描述分子冷冻EM与结晶学结合使用的出版物急剧增长，它们揭示了机械生物学特别有趣的特征。分子冷冻EM填补了高分辨率细胞学EM和结晶学之间的独特领域。通过检查没有固定在晶格中的样品，可以表征无法通过结晶学表征的大分子组装的动态特征，并确定太大或太不均匀而无法结晶的物种的亚纳米结构。一种非常有效的策略是通过结晶学或核磁共振来确定大分子络合物组分的高分辨率结构，然后基于对完整组织的低温电子显微镜重建来构建伪原子分辨率模型。在目前高压FEG仪器可以达到的分辨率下，这种对低温电磁场密度的建模精度非常高。这项申请要求美国国立卫生研究院为一台300KV电子显微镜提供部分支持，该显微镜配备了一个场发射枪、一个氦低温工作台和一个4k×4k的慢扫描电荷耦合器件。该仪器将填补Torrey Pines台地上原子到细胞结构研究连续体的空白，该连续体是由参与机构、加州大学圣地亚哥分校、伯纳姆研究所和斯克里普斯研究所产生的世界级生物科学的支柱。建议使用该仪器进行高分辨率工作的项目包括分子马达、膜蛋白及其相互作用、控制细胞间通信的复合体以及病毒的组装和成熟。此外，为高通量EM数据采集开发的软件和硬件将在要求的显微镜上实施，因为它是完善的。这些都是资金充足的项目，受益于中等分辨率研究，并将在新技术可用时立即使用。我们预计，Torrey Pines Mesa的广泛项目将发现这项技术有用，因为目前没有直接参与分子低温EM的研究人员被介绍到从使用它产生的结果。

DESCRIPTION (provided by applicant): Molecular electron cryo-microscopy (cryoEM) is at the same stage of development today as macromolecular x-ray crystallography was in 1980. We are entering an exponential growth phase of this technology accompanied by significant expansion in the number of practitioners and applications. Publications describing the utilization molecular cryoEM in combination with crystallography have grown dramatically and they have revealed exceptionally interesting features of mechanistic biology. Molecular cryoEM fills a unique niche between high resolution cytological EM and crystallography. Examining specimens not immobilized in a crystal lattice has allowed the characterization of dynamic features of macromolecular assemblies impossible to characterize by crystallography and to determine sub nanometer structures of species too large or too heterogeneous to be crystallized. A remarkably powerful strategy has been to determine high-resolution structures of the components of a macromolecular complex by crystallography or NMR followed by the construction of a pseudo atomic resolution model based on the cryoEM reconstruction of the intact organization. At resolutions currently achievable with high voltage FEG instruments, the precision of this modeling into cryoEM density is exceptionally high. This application requests partial support from NIH for a 300KV electron microscope equipped with a field emission gun, a helium cryo stage and a 4k by 4k slow scan CCD. This instrument will fill a gap that exists on the Torrey Pines Mesa in the atoms to cells continuum of structural studies that is the backbone of the world-class biological science produced by the participating institutions; University of California, San Diego; The Burnham Institute and The Scripps Research Institute. Projects proposed for high resolution work with this instrument include molecular motors, membrane proteins and their interactions, complexes controlling intercellular communication and virus assembly and maturation. In addition, software and hardware developed for high throughput EM data collection will be implemented on the requested microscope as it is perfected. These are all well funded programs that have benefited from moderate resolution studies and will immediately utilize the new technology when it is available. We anticipate that a broad range of projects from the Torrey Pines Mesa will find this technology useful as investigators not currently directly involved with molecular cryoEM are introduced to results emerging from its use.