AN ELECTRON MICROSCOPE FOR THE STUDY OF COMPLEX BIOLOGICAL ASSEMBLIES

这个子项目是利用由NIH/NCRR资助的共享仪器赠款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是用于拨款的，而不一定是用于调查人员的机构。描述(申请人提供)：低温电子显微镜(Cryo-EM)可用于单颗粒和断层扫描的三维重建，是研究大分子组件和细胞细胞器的基本工具。一般来说，生物大分子组件(如病毒受体复合体和多形性病毒)不能结晶，因为它们不能有效地填充到晶格中，或者因为它们只有短暂的时间存在。冷冻电子断层扫描(Cryo-ET)的最新进展使小细胞(即直径为1微米的细胞)在其完全水合的自然状态下能够进行三维成像。当冷冻-ET与从高压冷冻制备的样品中冷冻水合物质的冷冻切片相结合时，可以在更大的生物样品上收集三维信息。这些电子显微镜技术填补了X射线结晶学和光学显微镜之间100-5000安培的范围。低温电子显微镜研究的标本在生理相关的缓冲溶液中玻璃化，重建的结构与自然结构非常接近。也可以确定不同功能状态的结构来表征生物系统的完整动态过程。在普渡，我们在结合低温EM和结晶学方面拥有丰富的经验。此外，为了表彰我们的一些成就，我们即将入住一座专为结构生物学而建的新大楼。我们将把我们目前的两台场发射枪传输电子显微镜转移到新设施，包括一台进入计划中的生物安全遏制设施，以研究危险的病毒病原体。放置在安全壳中的显微镜将受到限制，再加上过去五年期间显微镜使用量的增加，将需要购买一台新仪器。为此，我们希望购买一台最先进的300KV场发射枪仪器，该仪器具有能够在液氦温度下保持样品的工作台。这样的工具将允许我们在一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很容易识别。该仪器还将允许我们以大约50A的分辨率对缺乏结构同质性的多形性病毒和细胞器进行断层扫描研究。

This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. 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.