Transmission Electron Microscope

描述（由申请人提供）：分子电子冷冻显微镜（cryoEM）与1980年的大分子x射线晶体学处于相同的发展阶段。我们正在进入这项技术的指数级增长阶段，伴随着从业人员和应用程序数量的显著增长。描述分子低温电镜与晶体学结合使用的出版物急剧增长，它们揭示了机制生物学的异常有趣的特征。分子低温电镜在高分辨率细胞学电镜和晶体学之间填补了一个独特的利基。检查未固定在晶格中的样品，可以表征大分子组装的动态特征，这是晶体学无法表征的，也可以确定太大或太不均匀而无法结晶的亚纳米结构。一个非常强大的策略是通过晶体学或核磁共振确定大分子复合物组分的高分辨率结构，然后基于完整组织的低温电镜重建构建伪原子分辨率模型。在目前使用高压FEG仪器可实现的分辨率下，这种模拟到低温em密度的精度非常高。本申请要求美国国立卫生研究院部分支持配备场发射枪、氦冷冻级和4k × 4k慢扫描CCD的300KV电子显微镜。这台仪器将填补托里松台地在原子到细胞连续结构研究方面存在的空白，这种结构研究是各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。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、控制细胞间通讯的复合物以及病毒组装和成熟。此外，为高通量电磁数据采集而开发的软件和硬件将在要求的显微镜上实现，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，并将立即利用新技术。我们预计，Torrey Pines Mesa的广泛项目将发现这项技术很有用，因为目前没有直接参与分子低温技术的研究人员将被介绍到其使用产生的结果。

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.