goCTF: Geometrically optimized CTF determination for single-particle cryo-EM
goCTF: Geometrically optimized CTF determination for single-particle cryo-EM
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DOI：
10.1016/j.jsb.2018.11.012
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发表时间：
2019-01-01
影响因子：
3
通讯作者：
Su, Min
中科院分区：
文献类型：
--
作者：
Su, Min
优选的颗粒取向是单颗粒低温电子显微镜（cryo-EM）中反复出现的问题。已尝试通过倾斜的独立于样本的方法来增加粒子方向覆盖范围，从而最大限度地减少各向异性三维（3D）重建。然而，焦点梯度是阻碍倾斜应用成为单粒子冷冻电镜普遍应用的一个关键问题。本研究描述了一种新开发的几何优化方法 goCTF，可以可靠地确定全局焦点梯度。应用一种从信号保留功率谱的扇区确定对比度传递函数（CTF）参数的新策略来提高可靠性。随后，以迭代方式进行基于每粒子的局部焦点细化，以进一步提高散焦精度。还提出了使用标准差离焦图和拟合优度热图的新颖诊断方法，以在 3D 精修之前评估 CTF 拟合质量。在一项基准研究中，goC1P 处理了已发表的流感血凝素三聚体单粒子冷冻电镜数据集，该数据集是在 40 度标本倾斜条件下收集的。生成的 3D 重建图分辨率从 4.1 埃提高到 3.7 埃。 goCTF程序基于CTFFIND4的开源代码构建，采用一致的用户界面，易于使用。
Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refmement. In a benchmark study, goC1P processed a published single particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 angstrom to 3.7 angstrom resolution. The goCTF program is built on the open source code of CTFFIND4, which adopts a consistent user interface for ease of use.