AN ELECTRON MICROSCOPE FOR THE STUDY OF COMPLEX BIOLOGICAL ASSEMBLIES

该子项目是利用由NIH/NCRR资助的共享仪器补助金提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是资助机构，不一定是研究者的机构。描述（由申请人提供）：冷冻电子显微镜（cryo-EM）可用于单颗粒和层析三维重建，是研究大型大分子组装和细胞器的重要工具。一般来说，生物大分子组合物（如病毒-受体复合物和多形性病毒）不能结晶，因为它们不能有效地装入晶格中，或者因为它们的存在时间很短。低温电子断层扫描（cryo-ET）的最新进展允许在完全水化的天然状态下对小细胞（即直径<1瓮）进行三维成像。当cryo-ET与高压冷冻制备的样品的冷冻水化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介于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，重建的结构与原始结构密切相关。还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，我们在结合低温电镜和晶体学方面拥有丰富的经验。此外，为了表彰我们的一些成就，我们将被安置在一栋专门用于结构生物学的新楼里。我们将把现有的两台场发射枪透射电子显微镜搬到新设施，其中一台将搬进计划中的生物安全控制设施，用于研究危险的病毒病原体。安置在容器内的显微镜进入将受到限制，再加上过去五年期间显微镜使用的增加，将有必要购置一台新仪器。为此，我们希望购买最先进的300千伏场发射枪仪器，该仪器具有能够在液氦温度下维持样品的级。这样的仪器将允许我们在一些有利的情况下将重建推到5a或更好。在这样的分辨率下，蛋白质和核酸的二级结构特征很容易识别。该仪器也将允许我们进行层析研究，在大约50分辨率的多形性病毒和细胞器缺乏结构的同质性。

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.