In-Focus Phase Contrast for Cryo-EM

用于冷冻电镜的聚焦相差

• 批准号: 8537936
• 负责人: KENNETH H DOWNING
• 金额: $ 58.86万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537936
• 关键词: 3-Dimensional; Address; Binding; Biochemistry; Biological; Biological Testing; Biomedical Research; C-terminal; Cells; Cellular biology; Charge; Chemicals; Complex; Computers; Data Quality; Data Set; Dependence; Development; Device Designs; Devices; Due Process; Effectiveness; Electrodes; Electron Microscope; Electron Microscopy; Electrons; Electrostatics; Evaluation; Exhibits; Fourier Transform; Frequencies; Goals; Grant; Heterogeneity; High temperature of physical object; Hybrids; Ice; Image; Ions; Laboratories; Lasers; Learning; Medical Research; Methods; Microtubules; Modeling; Molecular; Molecular Conformation; Molecular Weight; Noise; Phase; Proteins; Radial; Relative (related person); Research; Research Project Grants; Resolution; Rest; Sampling; Signal Transduction; Specimen; Streptavidin; Structure; Sulfite reductase; Surface; Tail; Technology; Testing; Time; Tobacco Mosaic Virus; Tomogram; Tongue; Tube; Tubulin; Use Effectiveness; Work; base; design; flexibility; genetic regulatory protein; improved; macromolecular assembly; monolayer; novel; particle; protein complex; reconstruction; structural biology; tomography; tool; transmission process; voltage

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the effectiveness of using charging-free versions of two fundamentally different types of microfabricated phase-contrast devices, designed for use in the transmission electron microscope. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 200 kDa should be more easily identified and selected for computer processing, due to the improved contrast transfer function (CTF) at low spatial frequencies that is provided by in-focus phase contrast. Structural information at high resolution will no longer be corrupted or lost due to use of large values of defocus to achieve the needed amount of phase contrast. The improved image quality may result in improvement in the ability to detect structural (compositional) and conformational differences amongst particles in a structurally non-homogeneous sample. One type of device that will be evaluated is a microfabricated aperture that blocks a half-plane of the electron wave that is scattered at small angle, while at the same time allowing the rest of the scattered wave to contribute in the usual way to image formation. The second type of device is a novel, 2-electrode "electrostatic drift tube" design that applies a 90 degree phase shift to the unscattered electrons relative to the scattered electrons. Our fabrication of charging-free devices will employ high-temperature annealing and refinement of materials and devices, chemical and physical cleaning of surfaces, and the use of sensitive tools for detecting surface contamination. Fabrication methods will include traditional lithographic wet-fabrication as well as laser micromachining and focused-ion beam milling. The effectiveness of these devices will be determined from cryo-EM images of biological test specimens such as monolayer crystals of streptavidin and tobacco mosaic virus. Applications to current research will include studies on structural plasticity of microtubules and the interactions of various regulatory proteins with microtubules.

描述（由申请人提供）：拟议的研究的目的是确定使用两种基本不同类型的微型相对对比设备的无充电版本的有效性，该版本旨在用于传输电子显微镜。在生物医学研究中使用焦点内相的对比度的优点预计是：由于在低空间频率下改善的对比度传递函数（CTF），应更容易地识别出小至200 kDa的蛋白质复合物的图像，并选择用于计算机处理。 高分辨率的结构信息将由于使用大量散焦而不再被损坏或丢失，以实现所需的相比对比度。 改善的图像质量可能会提高检测结构（组成）和结构非均匀样品中颗粒之间的构象差异。一种将要评估的设备是一种微型孔径，该光圈阻塞了以小角度散射的电子波的半平面，同时允许其余的散射波以通常的图像形成方式贡献。第二种设备是一种新颖的2电极“静电漂移管”设计，该设计将90度相移相对于散射电子。我们对无充电设备的制造将采用高温退火和材料和设备的完善，表面的化学和物理清洁以及使用敏感工具来检测表面污染。制造方法将包括传统的光刻湿制作以及激光微加工和聚焦式束铣削。这些设备的有效性将取决于生物测试样品的冷冻EM图像，例如链霉亲和蛋白链球菌的单层晶体和烟草马赛克病毒。当前研究的应用将包括有关微管结构可塑性的研究，以及各种调节蛋白与微管的相互作用。