Three-Dimensional Reconstruction from Electron Micrographs of Randomly Oriented Macromolecules

随机取向大分子的电子显微照片的三维重建

• 批准号: 9515518
• 负责人: Charles Lawrence
• 金额: $ 44.39万
• 依托单位: Health Research Incorporated/New York State Department of Health
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-15 至 2001-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9515518&HistoricalAwards=false
• 关键词: Three; Dimensional; Reconstruction; Electron; Micrographs

Three-dimensional Reconstruction from Electron Micrographs of Randomly Oriented Macromolecules. Knowledge of the structure of macromolecules is essential to an understanding of their functions in biological systems. Many large macromolecules can only be analyzed by threedimensional electron microscopy. For single asymmetrical particles, in contrast to crystalline objects, resolutions achieved in three dimensions have been limited to 2-3 nm. The techniques proposed here will contribute to a fuller usage of information contained in a micrograph and are an important step toward achieving higher resolution and thus a better understanding of biological function. A method is proposed for three-dimensional reconstruction from electron micrographs of randomly oriented single macromolecules. Biological particles are radiation sensitive, and imaging must be done under low-dose conditions. Only techniques that combine information of many identical particles can overcome the limits imposed by radiation sensitivity. One of the most successful methods of this kind was SECReT (Single Exposure Conical Reconstruction Technique), developed by the P.I.. Programs for this method have been distributed to laboratories world wide and have been used for the determination of more than 15 structures over the last seven years. However, SECReT requires that all particles used in a reconstruction have the same orientation within the plane of the specimen. Deviations from this preferred orientation limit the resolution in three dimensions. Proposed here is a new method for determining the orientation of particles from their projections. This technique can be used for refining the orientation of projections used for SECReT and for the determination of projection angles of particles with unknown orientations. Successful development of this technique will lead to substantial reduction in experimental effort and increase the resolution achievable in three dimensions. The theory of Radon t ransforms is the basis for three-dimensional reconstruction algorithms. Most research has focused on the design of efficient inversion algorithms and only a few applications have made use of their potential as image processing tools. In this proposal Radon transforms are used as tools for simultaneous translational and rotational alignment of projections in three dimensions. Once the orientations of the projections are known a threedimensional reconstruction can be calculated. The alignment/reconstruction method will be combined with a Gibbs sampling technique for overcoming noise limitations and for image classification. Programs to assess the feasibility of the proposed technique have been written and first results have been published. The new technique will be applied to two objects, the calcium release channel from sarcoplasmic reticulum and the 50S ribosomal subunit from Escherichia coli. The two macromolecules were chosen because they exhibit features that are advantageous for testing the procedures, and because results at higher resolution will contribute to a better understanding of their function. The four-fold symmetry of the calcium release channel can be used to increase the signal to noise ratio, whereas the 50S ribosomal subunit is a globular structure with few protuberances and ideal for testing the limits of the applicability of this method.

从随机取向大分子的电子显微照片进行三维重建。 了解大分子的结构对于理解它们在生物系统中的功能至关重要。许多大的大分子只能通过三维电子显微镜进行分析。与晶体物体相比，对于单个不对称粒子，在三个维度上实现的分辨率仅限于 2-3 nm。这里提出的技术将有助于更充分地利用显微照片中包含的信息，并且是实现更高分辨率、从而更好地理解生物功能的重要一步。 提出了一种根据随机取向的单个大分子的电子显微照片进行三维重建的方法。 生物颗粒对辐射敏感，成像必须在低剂量条件下进行。只有结合许多相同粒子信息的技术才能克服辐射敏感性带来的限制。这种方法中最成功的方法之一是由 P.I. 开发的 SECReT（单次曝光圆锥重建技术）。该方法的程序已分发给世界各地的实验室，并在过去七年中用于确定超过 15 个结构。然而，SECReT 要求重建中使用的所有粒子在样本平面内具有相同的方向。偏离该首选方向会限制三个维度的分辨率。这里提出了一种根据粒子投影确定粒子方向的新方法。该技术可用于细化用于 SECReT 的投影方向以及确定未知方向粒子的投影角度。该技术的成功开发将大大减少实验工作量并提高三维分辨率。 Radon 变换理论是三维重建算法的基础。大多数研究都集中在高效反演算法的设计上，只有少数应用程序利用了它们作为图像处理工具的潜力。在本提案中，Radon 变换被用作三维投影同步平移和旋转对齐的工具。一旦知道投影的方向，就可以计算三维重建。对齐/重建方法将与吉布斯采样技术相结合，以克服噪声限制并进行图像分类。评估所提议技术的可行性的程序已经编写，并且第一个结果已经发布。 这项新技术将应用于两个对象，即肌浆网的钙释放通道和大肠杆菌的50S核糖体亚基。选择这两种大分子是因为它们表现出有利于测试程序的特征，并且因为更高分辨率的结果将有助于更好地理解它们的功能。钙释放通道的四重对称性可用于提高信噪比，而 50S 核糖体亚基是球状结构，几乎没有突起，非常适合测试该方法的适用性极限。