Cryo-EM 3D Reconstruction of Flexible Particles

柔性颗粒的冷冻电镜 3D 重建

• 批准号: 8182649
• 负责人: Hemant D Tagare
• 金额: $ 28.49万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8182649
• 关键词: Algorithms; Bayesian Analysis; Benchmarking; Cells; Code; Complex; Computer software; Data; Dicer Enzyme; Electron Microscopy; Environment; Enzymes; Freedom; Freezing; Heterogeneity; Human; Ice; Image; Individual; Macromolecular Complexes; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Negative Staining; Noise; Probability Theory; Process; Property; Pythons; RNA; RNA Interference Pathway; RNA Polymerase II; RNA Processing; Shapes; Signal Transduction; Simulate; Structural Models; Structure; Techniques; Writing; base; combat; cryogenics; expectation; flexibility; human DICER1 protein; image processing; novel strategies; particle; reconstruction; simulation; sound; three dimensional structure

DESCRIPTION (provided by applicant): Single-particle reconstruction (SPR) electron microscopy is a powerful technique for obtaining the 3D structure of macromolecular complexes in near-native states. A major problem in SPR is that flexibility gives rise to multiple conformations when the macromolecular particles are frozen in vitreous ice. Traditional SPR assumes that macromolecular particles are rigid, and gives good reconstructions only when all particles have the identical structure. We propose to develop new reconstruction methods which eliminate the rigidity assumption altogether. These methods can reconstruct a continuously flexible structure. Two classes of methods are proposed: (a) methods for exploratory analysis which detect the presence of structural conformational change and objectively determine the most dominant modes of flexibility, and (b) post- exploratory flexible particle reconstruction methods which reconstruct in detail the identified modes of flexibility. These methods are based on probability theory and preliminary results with actual data show that they offer significantly higher signal-to-noise ratio (SNR) reconstructions even when the data SNR is as low as -25db. Further, our methods provide a simple interpretation of particle flexibility. We will develop these algorithms and write software modules for incorporation in the SPARX cryo-EM processing environment. As a first application, we will use the software to process negative-staining and cryo-EM images of the human Dicer RNA-processing enzyme. As positive and negative controls, we will also reconstruct the structure of RNA Polymerase II and GroEL. PUBLIC HEALTH RELEVANCE: Electron microscopy can provide detailed pictures of large molecular complexes from cells. Unfortunately, when the complexes are flexible, they take on different shapes at the instant they are frozen for imaging; in this case the reconstructions are blurred, because they are formed as averages from images of many individual complexes. We propose to develop a new approach for mathematically modeling the flexibility and allowing high-quality reconstructions to be made from flexible molecules such as human Dicer.

描述（由申请人提供）：单粒子重建（SPR）电子显微镜是一种用于获得接近天然状态的大分子复合物的 3D 结构的强大技术。 SPR 的一个主要问题是，当大分子颗粒冻结在玻璃冰中时，灵活性会产生多种构象。传统的SPR假设大分子颗粒是刚性的，只有当所有颗粒具有相同的结构时才能给出良好的重建。我们建议开发新的重建方法来完全消除刚性假设。这些方法可以重建连续灵活的结构。提出了两类方法：（a）探索性分析方法，检测结构构象变化的存在并客观地确定最主要的灵活性模式，以及（b）探索后柔性粒子重建方法，详细重建所识别的模式的灵活性。这些方法基于概率论，实际数据的初步结果表明，即使数据 SNR 低至 -25db，它们也能提供显着更高的信噪比 (SNR) 重建。此外，我们的方法提供了粒子灵活性的简单解释。我们将开发这些算法并编写软件模块以合并到 SPARX 冷冻电镜处理环境中。作为第一个应用，我们将使用该软件处理人类 Dicer RNA 处理酶的负染色和冷冻电镜图像。作为阳性和阴性对照，我们还将重建RNA聚合酶II和GroEL的结构。 公共健康相关性：电子显微镜可以提供细胞大分子复合物的详细图片。不幸的是，当复合物具有柔性时，它们在冷冻成像时会呈现不同的形状。在这种情况下，重建是模糊的，因为它们是由许多单独复合体的图像的平均值形成的。我们建议开发一种新方法，对灵活性进行数学建模，并允许从人类 Dicer 等柔性分子中进行高质量的重建。