Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, 237 BSRB, 615 Charles E. Young Dr. S., Los Angeles, CA 90095-7364, USA.
J Struct Biol. 2011 Sep;175(3):253-63. doi: 10.1016/j.jsb.2011.05.004. Epub 2011 May 24.
To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors--in both cryoEM image acquisition and 3D reconstruction--that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved 'characteristic surfaces' in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.
为了将大生物复合物的冷冻电子显微镜（cryoEM）推向原子分辨率，在 cryoEM 图像采集和 3D 重建中，一些在低分辨率下可能被忽略的因素变得非常重要。在这里，我们对四个限制因素进行了彻底的分析：（a）电子束倾斜，（b）离焦值的不准确确定，（c）颗粒中的焦点梯度，以及（d）对于大颗粒，电子的动态（多次）散射。我们还提出了应对这些因素的策略：（a）通过保持平行照明和使用无彗差对准程序，可以减少电子束倾斜的发散和方向倾斜分量。此外，通过使用球差校正器，可以消除所有电子束倾斜分量，包括螺旋倾斜。（b）通过在高电子剂量下对目标区域附近的区域成像并测量电子束倾斜引起的图像位移，可以获得更准确的离焦值测量。（c）在 cryoEM 图像的傅里叶变换中，每个已知的傅里叶系数都是 3D 结构中两个傅里叶系数的和，这两个傅里叶系数位于 3D 傅里叶空间中的两个弯曲的“特征曲面”上。我们描述了一种简单的基于模型的迭代方法，可以在两个特征曲面上恢复这两个傅里叶系数。（d）通过传递函数的反卷积可以校正动态散射的影响。这些分析和我们提出的策略为未来以原子分辨率为目标的 cryoEM 重建实验设计提供了有用的指导。
