In-focus Phase Contrast for Cryo-EM

描述(申请人提供)：拟议研究的目标是确定是否可以开发出在生物大分子的电子显微镜中使用静电相衬孔(“四分之一波片”)进行聚焦相衬的技术。在生物医学研究中使用聚焦相位对比度的优势预计是： 小到250 kDa的蛋白质复合体的图像应该更容易识别和选择 计算机处理，由于改进了低空间频率下的对比度传递函数(CTF) 这是由聚焦相位对比度提供的。 分辨率为0.8 nm的结构信息将不再由于使用大型 散焦值，以达到所需的相位对比度。 改进的图像质量可以导致检测结构成分的能力的改进) 以及非均匀样品中颗粒之间的构象差异。 这项拟议研究的最终目标是确定是否通过 相衬孔径可用于以0.8纳米的分辨率获得小至250 kDa的多蛋白质复合体的可解释的三维重建。这项工作是基于一种新颖的双电极“漂移管”设计，这种设计在亚微米尺寸的制造方面具有一些优势。根据我们的初步研究，我们现在建议将该器件与经过修改的电子光柱相结合，以便在电子衍射图的大小和微加工孔径的大小之间提供所需的匹配。我们将使用分辨率“g”和零频率的傅里叶变换幅度比[即F(G)/F(0)]来定量评估光圈充电对高分辨率对比度传递的影响，然后我们将使用这种定量分析来评估避免不必要充电的替代方法的有效性 该设备的。当我们证明该设备可以用于在~1/(30 Nm)到1/(0.8 nm)之间产生几乎平坦的CTF时，我们将自动化使用它进行低剂量EM所需的所有步骤。这个 自动化的功能是确保对焦相衬不会比基于散焦的相衬更难使用。此外，我们还将测试不同大小的多蛋白质复合体的聚焦相位对比性能，以确定是否有可能获得分辨率为0.8 nm的粒子的三维重建，这些粒子的分辨率高达250 kDa。

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.