In-focus Phase Contrast for Cryo-EM

用于冷冻电镜的聚焦相差

• 批准号: 7683279
• 负责人: ROBERT M GLAESER
• 金额: $ 70.06万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7683279
• 关键词: 3-Dimensional; Adopted; Automation; Biological; Biological Assay; Biomedical Research; Boxing; Charge; Collection; Computers; Data; Devices; Dimensions; Dose; Due Process; Effectiveness; Electrodes; Electron Microscopy; Electrons; Electrostatics; Ensure; Fourier Transform; Frequencies; Goals; Image; Methods; Microscope; Molecular Weight; Multiprotein Complexes; Optics; Pattern; Phase; Research; Research Design; Research Project Grants; Resolution; Sampling; Technology; Testing; Tube; Work; base; design; improved; lens; macromolecule; novel; particle; performance tests; prevent; protein complex; reconstruction

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine whether technology can be developed to use an electrostatic phase-contrast aperture ("quarter-wave plate") for in-focus phase contrast in electron microscopy of biological macromolecules. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 250 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 a resolution of 0.8 nm will no longer be corrupted or lost due to use of large value 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 non-homogeneous sample. The final goal for the proposed research is to determine whether image-data collected with the help of a phase-contrast aperture can be used to obtain interpretable, three-dimensional reconstructions of multiprotein complexes as small as 250 kDa at a resolution of 0.8 nm. This work is based upon a novel, 2-electrode "drift tube" design that has some advantages in terms of fabrication at sub-micrometer dimensions. Based on our preliminary studies, we now propose to combine this device with an electron optical column that is modified in order to provide the desired match between the size of the electron diffraction pattern and the size of the microfabricated aperture. We will use the ratio of Fourier-transform amplitudes at a resolution "g" and at zero frequency [i.e. F(g)/F(0)] to quantitatively evaluate the effect that charging of the aperture has on contrast transfer at high resolution, and we will then use this quantitative assay to evaluate the effectiveness of alternative methods to avoid unwanted charging of the device. When we have demonstrated that the device can be used to produce a nearly flat CTF between ~1/(30 nm) and 1/(0.8 nm), we will automate all steps needed to use it for low-dose EM. The function of automation is to ensure that in-focus phase contrast is no more difficult to use than is defocus based phase contrast. In addition, we will test the performance of in-focus phase contrast on multiprotein complexes of various sizes in order to determine whether it is possible to obtain 3-D reconstructions at a resolution of 0.8 nm for particles as "small" as 250 kDa.

描述（由申请人提供）：拟议的研究的目的是确定是否可以开发技术来使用静电相对比孔径（“四分之一波板”）在生物大分子的电子显微镜中进行对焦相对比。在生物医学研究中使用焦点内相对比的优势预计将是： 应更轻松地识别和选择蛋白质复合物的图像至250 kDa的图像 由于在低空间频率下的对比度传输函数（CTF），计算机处理 这是由焦点阶段对比提供的。 由于使用大型，分辨率为0.8 nm的结构信息将不再损坏或丢失 散焦的价值以实现所需的相比量。 提高的图像质量可能会提高检测结构组成的能力） 非均匀样品中颗粒之间的构象差异。 拟议研究的最终目标是确定是否借助于 相对比孔径可用于在0.8 nm的分辨率下对小至250 kDa的多蛋白络合物的可解释的三维重建。这项工作基于一种新颖的2电极“漂移管”设计，该设计在亚微米尺寸的制造方面具有一些优势。基于我们的初步研究，我们现在建议将该设备与电子光柱相结合，该设备经过修改，以提供电子衍射图和微型孔径大小之间的所需匹配。我们将在分辨率“ G”和零频率下使用傅立叶变换振幅的比率[即f（g）/f（0）]定量评估孔径充电对高分辨率的对比度转移的效果，然后我们将使用此定量测定来评估替代方法的有效性，以避免不需要的充电。 设备。当我们证明该设备可用于生成〜1/（30 nm）和1/（0.8 nm）之间的几乎平坦的CTF时，我们将自动化使用它用于低剂量EM所需的所有步骤。这 自动化的功能是确保与基于散焦的相位造影剂相比，焦点相比的使用并不难。此外，我们将测试各种大小的多蛋白复合物上的聚焦相对比度的性能，以确定是否有可能以“小”至250 kDa的粒子分辨率以0.8 nm的分辨率获得3-D重建。