An Electron Microscope for the study of Complex Biological Assemblies

描述（由申请人提供）：冷冻电子显微镜（cryo-EM）可用于单粒子和断层扫描三维重建，这是研究大分子组装体和细胞器的基本工具。通常，生物大分子组装体（例如，病毒-受体复合物和多形病毒）不能结晶，因为它们不能有效地填充到晶格中，或者因为它们仅具有短暂的暂时存在。冷冻电子断层扫描（cryo-ET）的最新进展允许对处于完全水合的天然状态的小细胞（即直径<1 μ m）进行三维成像。当冷冻ET与使用高压冷冻制备的样品的冷冻水合材料的冷冻切片相结合时，可以在更大的生物标本上收集三维信息。这些电子显微镜技术填补了X射线晶体学和光学显微镜之间的100-5000 A尺度范围。冷冻电镜研究的标本在生理相关的缓冲溶液中玻璃化，重建密切代表天然结构。也可以确定不同功能状态的结构，以表征生物系统的完整动态过程。在普渡大学，我们在结合cryo-EM和晶体学方面有着丰富的经验。此外，为了表彰我们的一些成就，我们将被安置在一个专门用于结构生物学的新建筑中。我们将把目前的两台场发射枪透射电子显微镜搬到新设施，其中一台将进入计划中的生物安全防护设施，以研究危险的病毒病原体。位于安全壳内的显微镜将受到限制，加上过去五年中显微镜的使用量增加，将需要购置一台新仪器。为此，我们希望购买一个国家的最先进的300千伏场发射枪仪器与阶段能够保持在液氦温度的标本。这样的仪器将使我们能够在某些有利的情况下将重建推到5 A或更好。在这样的分辨率下，蛋白质和核酸的二级结构特征变得容易识别。该仪器还将使我们能够对缺乏结构均一性的多形性病毒和细胞器进行约50 A分辨率的断层扫描研究。

DESCRIPTION (provided by applicant): Cryo-electron microscopy (cryo-EM) can be used for single particle and tomographic three- dimensional reconstructions, essential tools for the study of large macromolecular assemblies and cellular organelles. In general, biological macromolecular assemblies (e.g., virus-receptor complexes and pleomorphic viruses) cannot be crystallized because of their inability to pack efficiently into a crystal lattice or because they only have short-lived temporal existence. Recent advances in cryo-electron tomography (cryo-ET) allow for the three-dimensional imaging of small cells (i.e. <1 urn in diameter) in their fully hydrated, native state. When cryo-ET is combined with cryo-sectioning of frozen-hydrated material from samples prepared using high-pressure freezing, three-dimensional information can be gathered on even larger biological specimens. These electron microscopy techniques fill the 100-5000 A scale range between X-ray crystallography and optical microscopy. The specimens for cryo-EM studies are vitrified in physiologically relevant buffer solutions and the reconstructions closely represent the native structures. Structures of the different functional states can also be determined to characterize complete dynamic processes of biological systems. At Purdue, we have had extensive experience in combining cryo-EM and crystallography. Furthermore, in recognition of some of our accomplishments, we are about to be housed in a new building exclusively for Structural Biology. We will move both of our current field-emission gun transmission electron microscopes to the new facility, including one into the planned bio-safety containment facility to study hazardous viral pathogens. The microscope located in containment will have restricted access, and when combined with the increase in use of the microscopes over the past five-year period, will necessitate the acquisition of a new instrument. For this we wish to purchase a state-of-the-art 300 kV field emission gun instrument with a stage able to maintain specimens at liquid helium temperatures. Such an instrument would allow us to push reconstructions in some favorable cases to 5 A or better. At such resolutions, secondary structural features of proteins and nucleic acids become easily recognizable. The instrument would also allow us to perform tomographic studies at about 50 A resolution on pleomorphic viruses and cell organelles that lack structural homogeneity.