CLASSIFICATION AND AVERAGING OF SINGLE PARTICLES IN CRYO-ET

Cryo-ET 中单个颗粒的分类和平均

• 批准号: 7598618
• 负责人: MICHAEL F. SCHMID
• 金额: $ 4.89万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598618
• 关键词: 3-Dimensional; Accounting; Cells; Classification; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Collection; Electron Microscope; Electrons; Fourier Transform; Funding; Grant; Institution; Lead; Maps; Methods; Microscope; Modeling; Molecular Conformation; Noise; Numbers; Operative Surgical Procedures; Pattern; Problem Solving; Range; Relative (related person); Research; Research Personnel; Resolution; Resources; Rotation; Signal Transduction; Simulate; Sorting - Cell Movement; Source; Specimen; Structure; Subgroup; Tomogram; United States National Institutes of Health; particle; reconstruction; size; success; three-dimensional modeling; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Electron cryo-tomography has advantages for studying large objects having pleomorphic structures. However, tomography suffers from the "missing wedge" problem due to the limited tilts of specimens in the microscope column. Methods are being developed to sort out objects of the same symmetry, conformation or size by cross-correlation and then to average the subgroups separately in order to enhance the signal to noise ratio and thus the resolution. It is desirable to cross-correlate and average 3D volumes corresponding to particles isolated from a tomographic reconstruction. This is useful either to characterize the contents of a cell, or to develop an unbiased 3D model or models if the symmetry, size or conformation of the particles is uncertain. Determining the relative orientations of the particles is crucial to the success of this operation. However, we encountered a difficulty in these comparisons having to do with the geometry of data collection for tomographic reconstructions. Because a tilt angle range of ¿90¿ cannot be achieved in the electron microscope, central sections in Fourier space for tilts higher than 60-70¿ are missing, leading to the problem commonly referred to as the missing wedge in Fourier space. After extraction from the tomogram and before applying any rotations, the orientation of the missing wedge in Fourier space is identical for each particle/volume. However, the orientation of the particles themselves is random with respect to each other and must be searched for in 3D rotation space. Because of the strong signal from the pattern of the missing wedge, the cross-correlation map may merely lead to the alignment of the missing wedges, which is certainly not correct. This problem does not occur to the same extent in cases where an isotropic starting model is available and used for 3-D alignment. We solved this problem by understanding the relevant issues in reciprocal space. As two 3-D volumes in Fourier space, each containing a missing wedge, are rotated against each other in the cross-correlation search, and one volume is multiplied by the complex conjugate of the other, zeros are generated. They occur when the missing-data region for one of the particles is multiplied by data in the other particle, and the number of such zeros changes at each rotation angle increment in the search. To minimize the effect of the missing wedge on the overall scale of the 3-D cross-correlation map, we computed the number of non-zero terms after the complex multiplication in Fourier space for each relative 3-D orientation in the space searched for the two particles, and scaled the 3-D cross-correlation map at that orientation search angle by the reciprocal of this number. This correction factor is approximately right because the total power in the Fourier transform of a cross-correlation function influences the values of the subsequent cross-correlation map. This normalization was good enough to allow the true proper relative orientation of the particles to be determined as demonstrated from our simulated data with missing wedge (M.F. Schmid, unpublished). We also developed methods for accounting for the missing wedge when averaging particles together. Our methods lead to more accurate averages.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 电子冷冻术具有研究具有多态结构的大物体的优势。但是，由于显微镜柱中的样品倾斜有限，断层扫描遭受了“缺失楔形”问题。正在开发方法来通过互相关来整理相同的对称，会议或大小的对象，然后平均分别进行亚组，以增强信号与噪声比，从而提高噪声比，从而提高分辨率。 需要交叉且平均的3D体积，对应于从断层扫描重建中分离出的粒子。这对于表征细胞的内容很有用，也可以开发无偏的3D模型，或者如果颗粒的对称性，大小或构象不确定，则很有用。 确定颗粒的相对方向对于此操作的成功至关重要。但是，在这些比较中，我们遇到了与层析成像重建的数据收集几何形状有关的困难。因为在电子显微镜中无法实现»90»的倾斜角范围，因此缺失了高于60-70的倾斜空间中的中央部分，导致该问题通常被称为傅立叶空间中缺失的楔形。从断层图中提取并施加任何旋转之前，对于每个粒子/体积，在傅立叶空间中缺失的楔形的方向是相同的。但是，粒子本身的方向相对于彼此是随机的，必须在3D旋转空间中搜索。由于缺失楔形的模式的强烈信号，互相关图可能仅导致缺失的楔形物的对齐，这肯定是不正确的。在各向同性启动模型可用并用于3-D对齐的情况下，此问题并不是同样的程度。 我们通过了解相互空间中的相关问题解决了这个问题。由于在傅立叶空间中有两个3-D体积（每个包含缺失的楔形物）在交叉相关搜索中相互旋转，并且一个体积由另一个复杂的共轭物多重，因此生成了零。当将其中一个粒子的缺失数据区域乘以另一个粒子中的数据时，并且此类零在搜索中每个旋转角度增加时的数量变化时就会发生。为了最大程度地减少缺失的楔形物对3-D互相关图的整体规模的影响，我们计算了傅立叶空间中复杂乘法后的非零项的数量，用于搜索两个粒子的空间中的每个相对3-D方向，并按照该数字的往分将3-D交叉相关图缩放。该校正因子大约正确，因为互相关函数的傅立叶变换中的总功率会影响随后的互相关图的值。这种归一化足以使粒子的真正正确相对取向被确定，如我们的模拟数据所证明的那样（M.F. Schmid，未发表）。 我们还开发了用于将颗粒平均在一起时丢失楔形的方法。我们的方法导致更准确的平均值。