High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy.
High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy.
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DOI：
10.1016/j.ultramic.2013.06.004
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发表时间：
2013-12
期刊：
影响因子：
2.2
通讯作者：
Henderson, Richard
中科院分区：
文献类型：
--
作者：
Chen, Shaoxia;McMullan, Greg;Faruqi, Abdul R.;Murshudov, Garib N.;Short, Judith M.;Scheres, Sjors H. W.;Henderson, Richard
单颗粒冷冻电子显微镜（cryoEM）确定三维（3D）结构包括计算初始3D模型，随后对单个颗粒图像的取向确定以及所得的3D图谱进行大量迭代改进。由于图像在高分辨率下噪声比信号多得多，这就有可能在3D图谱中增强噪声，从而对所达到的分辨率产生错误印象。最终图谱在其极限分辨率下的信号与噪声之间的平衡取决于图像处理过程，且不易预测。冷冻电镜领域越来越意识到如何避免这种过度拟合和对分辨率的高估。同样，人们一直不愿使用两种主要的避免方法，因为它们给出的分辨率估计值较低，有些人认为这过于悲观。在此我们描述一种与任何图像处理协议都兼容的简单测试。该测试可以测量最终3D图谱中存在的信号量以及因过度拟合产生的噪声量。我们使用几种图像处理软件包将该方法应用于两组不同的β - 半乳糖苷酶的冷冻电镜图像。我们的步骤包括用来自相邻背景区域的傅里叶分量，或者通过对颗粒结构因子的相位进行简单随机化，来替换超出选定分辨率的初始颗粒图像堆栈的傅里叶分量。因此，这种替换的噪声具有与原始数据相同的频谱功率分布。比较使用实验数据获得的3D图谱的傅里叶壳层相关（FSC）图与替换了高分辨率噪声（HR - 噪声）的相同数据的FSC图，可以明确测量过度拟合的量以及进行相应的分辨率评估。即使存在大量过度拟合，也可以使用一个简单的公式从两条曲线计算出无偏的FSC。该方法与软件无关。因此，用户完全可以自由使用任何已有的方法或新的方法组合，只要同时进行HR - 噪声测试即可。将此过程应用于β - 半乳糖苷酶的冷冻电镜图像，结果表明过度拟合程度因处理过程而异，但在最佳情况下没有过度拟合，分辨率约为6 Å。（382字）
描述了一种验证生物结构的3D冷冻电镜图谱的新方法。
高分辨率噪声替换是一种测量单颗粒冷冻电镜中噪声过度拟合量的工具。
即使存在一些过度拟合，也能获得可靠的、无偏的分辨率估计。
通过冷冻电镜确定了分辨率约为6 Å的β - 半乳糖苷酶的结构。
Three-dimensional (3D) structure determination by single particle electron cryomicroscopy (cryoEM) involves the calculation of an initial 3D model, followed by extensive iterative improvement of the orientation determination of the individual particle images and the resulting 3D map. Because there is much more noise than signal at high resolution in the images, this creates the possibility of noise reinforcement in the 3D map, which can give a false impression of the resolution attained. The balance between signal and noise in the final map at its limiting resolution depends on the image processing procedure and is not easily predicted. There is a growing awareness in the cryoEM community of how to avoid such over-fitting and over-estimation of resolution. Equally, there has been a reluctance to use the two principal methods of avoidance because they give lower resolution estimates, which some people believe are too pessimistic. Here we describe a simple test that is compatible with any image processing protocol. The test allows measurement of the amount of signal and the amount of noise from overfitting that is present in the final 3D map. We have applied the method to two different sets of cryoEM images of the enzyme beta-galactosidase using several image processing packages. Our procedure involves substituting the Fourier components of the initial particle image stack beyond a chosen resolution by either the Fourier components from an adjacent area of background, or by simple randomisation of the phases of the particle structure factors. This substituted noise thus has the same spectral power distribution as the original data. Comparison of the Fourier Shell Correlation (FSC) plots from the 3D map obtained using the experimental data with that from the same data with high-resolution noise (HR-noise) substituted allows an unambiguous measurement of the amount of overfitting and an accompanying resolution assessment. A simple formula can be used to calculate an unbiased FSC from the two curves, even when a substantial amount of overfitting is present. The approach is software independent. The user is therefore completely free to use any established method or novel combination of methods, provided the HR-noise test is carried out in parallel. Applying this procedure to cryoEM images of beta-galactosidase shows how overfitting varies greatly depending on the procedure, but in the best case shows no overfitting and a resolution of ~6 Å. (382 words) A new method to validate 3D cryoEM maps of biological structures is described. High-resolution noise substitution is a tool to measure the amount of overfitting of noise in single particle cryoEM. A reliable, unbiased resolution estimation can be obtained even when some overfitting is present. Structure of beta-galactosidase at ~6 Å resolution is determined by cryoEM.
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DOI：
10.1016/j.str.2010.05.008
发表时间：
2010-07-14
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
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通讯作者：
Frank J
DOI：
10.1139/o10-009
发表时间：
2010-10-01
期刊：
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE
影响因子：
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作者：
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通讯作者：
Huber, Reuben E.
影响因子：
3
作者：
Crowther, RA;Henderson, R;Smith, JM
通讯作者：
Smith, JM
DOI：
10.1073/pnas.0913774107
发表时间：
2010-03-16
影响因子：
11.1
作者：
Cong, Yao;Baker, Matthew L.;Chiu, Wah
通讯作者：
Chiu, Wah
影响因子：
3
作者：
Elmlund, Dominika;Elmlund, Hans
通讯作者：
Elmlund, Hans