Acquisition of a 300KeV FEG Energy-Filtered Liquid Helium-Stage Electron Microscope for Three-Dimensional Analysis of Supramolecular and Cellular Structures

购买 300KeV FEG 能量过滤液氦级电子显微镜，用于超分子和细胞结构的三维分析

• 批准号: 0079441
• 负责人: David Agard
• 金额: --
• 依托单位: University of California-San Francisco
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0079441&HistoricalAwards=false
• 关键词: Acquisition; 300KeV; FEG; Energy; Filtered

A state-of-the-art Philips Tecnai F30 Helium Electron Microscope with an attached Gatan Imaging Energy Filter (GIF) and 2Kx2K CCD detector will be used for high-resolution biological imaging at both the atomic and cellular levels. Increasingly, the frontiers of cell biology focus on elucidating the control of cellular shape and organization, the nature and function of cellular organelles, and the role of complex protein machines. Such project areas also define a new frontier for structural biology where the goals are to determine not the structures of individual molecules, but structures of supramolecular complexes as large as entire cellular organelles. Embodied in this goal is a switch from the largely reductionist approach of the past to one that is, at its heart, integrative. Electron microscopy (EM) is uniquely poised to meet this challenge. Single particle reconstruction methods (SPR) hold the promise of near atomic resolution structures of very large complexes while Intermediate Voltage EM Tomography (EMT) provides the unique ability to integrate this information into the context of the whole cell. The combination of Intermediate voltage (300kV) and Field Emission Gun provide optimal imaging for both thin and thick samples. Liquid helium cooling of the sample significantly reduces the effects of beam damage as well as the completely redesigned tilting stage provides unprecedented sample stability and freedom from drift. Addition of an imaging energy filter will revolutionize thick-section imaging and improve cryo imaging by removing inelastic electron scatter. The large area CCD will provide optimal on-line digital image recording. The result will be a facility unique in the US. Goals are to reach 25 angstrom resolution on EMT reconstruction of samples 200nm thick and to reach 4-7 angstrom resolution from SPR of particles larger than 500 KDa. The combination of the capabilities of this microscope with automated data collection methods being developed, should enable near atomic resolution reconstruction of single macromolecular complexes to become a reality.This instrumentation will empower major advances in cell biology and polymer science. A core user group of scientists from UCSF, Stanford, Berkeley, as well as the more distant Harvard and U. North Carolina has substantial tomography expertise and focuses on fundamentally important problems in biology and polymer science. Projects range from the understanding mechanisms controlling actin and microtubule cytoskeletal organization, the structure of chromosomes and mitotic spindle, the structure of the transcriptional initiation complex, the organization of the neuromuscular junction, and the guiding principles of organization in complex plastics.The equipment will be in the existing Electron Microscope Laboratory in the UCSF Biochemistry Department and in August 2002 will be moved to the new Mission Bay campus. This facility is available to the entire campus as well as to outside users. A center for electron microscope tomography will act as a magnet to attract and train top students and postdocs. The UCSF programs in Biophysics and Cell Biology provide excellent opportunities for training graduate students and actively seek to attract minority students to campus.

将使用最先进的Philips Tecnai F30氦电子显微镜，附带Gatan成像能量过滤器（GIF）和2Kx 2K CCD探测器，在原子和细胞水平进行高分辨率生物成像。越来越多的细胞生物学的前沿集中在阐明细胞形状和组织的控制，细胞器的性质和功能，以及复杂蛋白质机器的作用。这些项目领域也为结构生物学定义了一个新的前沿，其目标不是确定单个分子的结构，而是确定整个细胞器大小的超分子复合物的结构。这一目标的核心是从过去的简化主义方法转向核心的综合方法。电子显微镜（EM）是唯一准备迎接这一挑战。单粒子重建方法（SPR）有望获得非常大的复合物的近原子分辨率结构，而中压EM断层扫描（EMT）提供了将此信息整合到整个细胞背景中的独特能力。中间电压（300 kV）和场发射枪的组合为薄和厚样品提供了最佳成像。样品的液氦冷却显著降低了光束损伤的影响，并且完全重新设计的倾斜载物台提供了前所未有的样品稳定性和免于漂移的自由。增加成像能量过滤器将彻底改变厚切片成像，并通过消除非弹性电子散射来改善冷冻成像。大面积CCD将提供最佳的在线数字图像记录。其结果将是一个独特的设施在美国。 目标是在200 nm厚的样品的EMT重建上达到25埃分辨率，并且从大于500 KDa的颗粒的SPR达到4-7埃分辨率。这种显微镜的功能与正在开发的自动数据收集方法相结合，应该能够使单个大分子复合物的近原子分辨率重建成为现实。这种仪器将使细胞生物学和聚合物科学取得重大进展。一个核心用户群的科学家来自加州大学旧金山分校，斯坦福大学，伯克利分校，以及更遥远的哈佛和美国。北卡罗来纳州拥有大量的断层扫描专业知识，并专注于生物学和聚合物科学中的重要问题。项目范围从理解控制肌动蛋白和微管细胞骨架组织的机制，染色体和有丝分裂纺锤体的结构，转录起始复合体的结构，神经肌肉接头的组织，以及复杂塑料组织的指导原则。该设备将位于加州大学旧金山分校生物化学系现有的电子显微镜实验室，并将于2002年8月搬迁新的使命湾校区该设施可供整个校园以及外部用户使用。一个电子显微镜断层扫描中心将像磁铁一样吸引和培养优秀的学生和博士后。UCSF的生物物理学和细胞生物学课程为培养研究生提供了极好的机会，并积极吸引少数民族学生进入校园。