CryoEM single particle reconstruction with a complex-valued particle stack.
CryoEM single particle reconstruction with a complex-valued particle stack.
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DOI：
10.1016/j.jsb.2023.107945
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发表时间：
2023-06
影响因子：
3
通讯作者：
Otwinowski, Zbyszek
中科院分区：
文献类型：
--
作者：
Bromberg, Raquel;Guo, Yirui;Borek, Dominika;Otwinowski, Zbyszek
关键词：
Cryoem中的单粒子重建（SPR）是一个图像处理任务，其精细层次结构从许多非常嘈杂的多帧图像开始。中介图像结构的有效表示对于保持计算易于管理至关重要。一种这样的中介结构称为粒子堆，包含预定义尺寸的平方盒中粒子的切割图像。通常，在粒子堆栈创建之前，通常对框图像来源的显微照片进行校正。但是，在此步骤中未考虑对比度传输函数（CTF）或其傅立叶变换点扩散函数（PSF）。从历史上看，粒子堆栈旨在用于大颗粒和更紧密的PSF，这是较低分辨率数据的特征。现在，该领域对较小的粒子进行分析和更高的分辨率，这些条件会导致更广泛的PSF，需要更大的填充和较慢的计算以整合每个粒子的信息。因此，应重新检查处理结构（例如粒子堆栈）的方法以优化数据处理。
在这里，我们建议将粒子堆叠的源图像用作复杂值的图像，其中CTF校正被隐式应用为图像的实际组成部分。我们可以通过将初始CTF校正对整个显微照片进行应用，并作为后续步骤执行盒式切口来实现它。我们完善并施加的最终CTF校正具有非常狭窄的PSF，因此从近似CTF校正的显微照片中切出颗粒不需要扩展缓冲，即分析过程中的盒子只需要足够大就可以包含粒子。出口波重建的傅立叶变换创造了具有复杂值的图像。这是在真实空间中考虑的复杂值图像，与标准SPR数据处理相反，其中复数仅在傅立叶空间中出现。显微照片概念的扩展提供了多个优点，因为粒子盒的大小可能很小，并且对于高分辨率重建至关重要的计算，例如Ewald球体校正，畸变细化和粒子特异性的散焦细化，可以在小型盒数据上执行。
Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with many very noisy multi-frame images. Efficient representation of the intermediary image structures is critical for keeping the calculations manageable. One such intermediary structure is called a particle stack and contains cut-out images of particles in square boxes of predefined size. The micrograph that is the source of the boxed images is usually corrected for motion between frames prior to particle stack creation. However, the contrast transfer function (CTF) or its Fourier Transform point spread function (PSF) are not considered at this step. Historically, the particle stack was intended for large particles and for a tighter PSF, which is characteristic of lower resolution data. The field now performs analyses of smaller particles and to higher resolution, and these conditions result in a broader PSF that requires larger padding and slower calculations to integrate information for each particle. Consequently, the approach to handling structures such as the particle stack should be reexamined to optimize data processing. Here we propose to use as a source image for the particle stack a complex-valued image, in which CTF correction is implicitly applied as a real component of the image. We can achieve it by applying an initial CTF correction to the entire micrograph first and perform box cutouts as a subsequent step. The final CTF correction that we refine and apply later has a very narrow PSF, and so cutting out particles from micrographs that were approximately corrected for CTF does not require extended buffering, i.e. the boxes during the analysis only have to be large enough to encompass the particle. The Fourier Transform of an exit-wave reconstruction creates an image that has complex values. This is a complex value image considered in real space, opposed to standard SPR data processing where complex numbers appear only in Fourier space. This extension of the micrograph concept provides multiple advantages because the particle box size can be small and calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particle-specific defocus refinement can be performed on the small box data.
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