Zhang Wei, Kimmel Marek, Spahn Christian M T, Penczek Pawel A
The University of Texas-Houston Medical School, Department of Biochemistry and Molecular Biology, Houston, TX 77030, USA.
Structure. 2008 Dec 10;16(12):1770-6. doi: 10.1016/j.str.2008.10.011.
Macromolecular structure determination by cryo-electron microscopy (EM) and single-particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. We describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set of its projections. We introduce a highly efficient reconstruction algorithm that is based on direct Fourier inversion and that incorporates correction for the transfer function of the microscope, thus extending the resolution limits of variance estimation. We also describe a validation method to determine the number of resampled volumes required to achieve stable estimate of the variance. The proposed bootstrap method was applied to a data set of 70S ribosome complexed with tRNA and the elongation factor G. The proposed method of variance estimation opens new possibilities for single-particle analysis, by extending applicability of the technique to heterogeneous data sets of macromolecules and to complexes with significant conformational variability.
通过冷冻电子显微镜（EM）和单颗粒分析确定大分子结构是基于成像分子具有相同结构这一假设。随着处理数据集规模的增大，很明显许多复合物以构象状态混合物的形式共存，或者包含柔性区域。我们描述了一种自展重采样技术的实现方法，该方法可对从一组投影重建的结构逐体素方差进行估计。我们引入了一种基于直接傅里叶反演的高效重建算法，该算法纳入了对显微镜传递函数的校正，从而扩展了方差估计的分辨率极限。我们还描述了一种验证方法，以确定实现方差稳定估计所需的重采样体积数量。所提出的自展方法应用于与tRNA和延伸因子G复合的70S核糖体数据集。所提出的方差估计方法通过将该技术的适用性扩展到异质大分子数据集以及具有显著构象变异性的复合物，为单颗粒分析开辟了新的可能性。
