Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps
Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps
复制标题
DOI：
10.1021/acs.jcim.9b01107
复制
发表时间：
2020-05-26
影响因子：
5.6
通讯作者：
Miyashita, Osamu
中科院分区：
文献类型：
--
作者：
Tiwari, Sandhya P.;Chhabra, Sahil;Miyashita, Osamu
低温电子显微镜（cryo-EM）单粒子分析在实现生物分子成像时的原子级分辨率方面已经取得了很大进展。为了在冷冻EM中获得最佳的三维（3D）结构，必须仔细考虑许多参数。在这里，我们解决了经常被忽视的像素大小参数，它描述了实验产生的图像的放大率。虽然努力完善和验证这一参数的分析cryo-EM实验数据，有没有系统的协议。由于像素大小参数可以有一个cryo-EM地图的分辨率和准确性的影响，并从它派生的原子分辨率的三维结构模型，我们提出了一个计算协议，以估计适当的像素大小参数。在我们的协议中，我们适合和完善原子结构对冷冻EM地图在多个像素大小。使用GOAP（广义取向依赖，全原子统计势）评分评估得到的拟合和细化结构，我们发现它比其他常用函数（如Molprobity和细化的相关系数）表现更好。最后，我们描述了该协议在检索适当的像素大小的几个例子，模拟数据的基础上酵母延伸因子2和实验数据从Gro-EL伴侣，β-半乳糖苷酶，和TRPV 1离子通道的功效。
Cryo-electron microscopy (cryo-EM) single-particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. To obtain the best possible three-dimensional (3D) structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution 3D structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (generalized orientation-dependent, all-atom statistical potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2 and experimental data from Gro-EL chaperone, beta-galactosidase, and the TRPV1 ion channel.