Transmission Electron Microscope

描述（由申请人提供）：分子电子冷冻显微镜（cryoEM）今天处于与1980年的大分子X射线晶体学相同的发展阶段。我们正在进入这项技术的指数增长阶段，伴随着从业人员和应用程序数量的显着扩张。描述利用分子cryoEM与结晶学相结合的出版物急剧增长，它们揭示了机械生物学的异常有趣的特征。分子cryoEM填补了高分辨率细胞学EM和晶体学之间的独特利基。检查不固定在晶格中的标本，允许表征的动态特性的大分子组装不可能表征的结晶学和确定亚纳米结构的物种太大或太异质结晶。一个非常强大的战略已经确定高分辨率的结构的组成部分的大分子复合物的结晶或NMR随后的伪原子分辨率模型的基础上的完整组织的cryoEM重建的建设。在目前用高压FEG仪器可实现的分辨率下，这种对cryoEM密度的建模的精度异常高。本申请要求NIH提供部分支持，用于配备场发射枪、氦冷冻台和4k × 4k慢扫描CCD的300 KV电子显微镜。该仪器将填补Torrey Pines梅萨上存在的原子到细胞连续结构研究的空白，该研究是参与机构产生的世界级生物科学的支柱;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。该仪器的高分辨率工作项目包括分子马达，膜蛋白及其相互作用，控制细胞间通讯和病毒组装和成熟的复合物。此外，为高通量EM数据收集开发的软件和硬件将在所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，受益于中等分辨率的研究，并将在新技术可用时立即使用。我们预计，广泛的项目从托里松梅萨将发现这项技术是有用的，因为调查人员目前没有直接参与分子cryoEM介绍了从其使用中出现的结果。

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.