Accurate determination of local defocus and specimen tilt in electron microscopy
Accurate determination of local defocus and specimen tilt in electron microscopy
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DOI：
10.1016/s1047-8477(03)00069-8
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发表时间：
2003-06-01
影响因子：
3
通讯作者：
Grigorieff, N
中科院分区：
文献类型：
--
作者：
Mindell, JA;Grigorieff, N
准确的离焦和倾斜参数的知识是必不可少的三维蛋白质结构的测定在高分辨率使用电子显微镜。我们提出了两个计算机程序，CTFFIND3和CTFTILT，确定散焦参数从图像的未倾斜的标本，以及散焦和倾斜参数从图像的倾斜标本，分别。这两个程序都使用了一个简单的算法，该算法将功率谱中可见的幅度调制与计算出的对比度传递函数（CTF）相匹配。使用低通滤波器计算功率谱中存在的背景。然后从原始功率谱中减去背景，仅允许CTF的振荡分量的拟合。CTFTILT通过测量图像上一系列位置处的散焦来确定样品倾斜参数，同时将它们约束到单个平面。我们测试了算法的二维晶体的图像，通过比较使用晶体学方法得到的结果。图像还包含来自碳支撑膜的对比度，其增加了CTF振荡的可见性。实验结果表明，该方法能够准确地确定像散，误差约为10 nm，而确定倾斜轴和倾斜角的误差分别约为20 °和P。进一步的测试是在冰中嵌入的单个蛋白质颗粒的图像上进行的，这些图像是从未倾斜或稍微倾斜的标本中记录的。由于缺少碳支撑膜，这些图像的CTF振荡的可见性降低。然而，测试结果表明，拟合过程能够确定图像散焦和倾斜角的误差分别约为100nm和60度。(C)2003 Elsevier Science（美国）。All rights reserved.
Accurate knowledge of defocus and tilt parameters is essential for the determination of three-dimensional protein structures at high resolution using electron microscopy. We present two computer programs, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from images of tilted specimens, respectively. Both programs use a simple algorithm that fits the amplitude modulations visible in a power spectrum with a calculated contrast transfer function (CTF). The background present in the power spectrum is calculated using a low-pass filter. The background is then subtracted from the original power spectrum, allowing the fitting of only the oscillatory component of the CTF. CTFTILT determines specimen tilt parameters by measuring the defocus at a series of locations on the image while constraining them to a single plane. We tested the algorithm on images of two-dimensional crystals by comparing the results with those obtained using crystallographic methods. The images also contained contrast from carbon support film that added to the visibility of the CTF oscillations. The tests suggest that the fitting procedure is able to determine the image defocus with an error of about 10 nm, whereas tilt axis and tilt angle are determined with an error of about 2degrees and P, respectively. Further tests were performed on images of single protein particles embedded in ice that were recorded from untilted or slightly tilted specimens. The visibility of the CTF oscillations from these images was reduced due to the lack of a carbon support film. Nevertheless, the test results suggest that the fitting procedure is able to determine image defocus and tilt angle with errors of about 100nm and 6degrees, respectively. (C) 2003 Elsevier Science (USA). All rights reserved.