A test-bed for optimizing high-resolution single particle reconstructions
A test-bed for optimizing high-resolution single particle reconstructions
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DOI：
10.1016/j.jsb.2008.04.005
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发表时间：
2008-07-01
影响因子：
3
通讯作者：
Potter, Clinton S.
中科院分区：
文献类型：
--
作者：
Stagg, Scott M.;Lander, Gabriel C.;Potter, Clinton S.
对于冷冻EM单粒子重建来说，生成分辨率类似于10埃的3D电子密度图已经成为一种常规，但分辨率为5埃或更高的图仍然是著名的事件。电子显微镜的分辨率优于2埃，因此不应成为限制因素；相反，分辨率的实际限制很可能来自样品制备方法、数据收集参数和数据分析程序的组合。在高度自动化的图像采集系统的帮助下，我们采用了一种系统的方法来优化影响单粒子重建分辨率的参数，并将其与关系数据库相结合，以跟踪所有处理参数。使用GroEL作为试验台，我们进行了一系列的3D重建，其中我们系统地改变了计算MAP时使用的粒子数量、显微镜的加速电压以及用于获取图像的电子剂量。我们还研究了排除不可接受或“不好的”粒子对最终3D贴图的影响的方法。使用相对标准的仪器(Tecnai F20，4K x 4K电荷耦合器件，侧入冷台)和完全自动化的方法，这些方法产生了名义分辨率为5.4埃(FSC0.5)的地图，其中二级结构清晰可辨，并且可以确定GroEL结构中一些α-螺旋的惯用手。(C)2008 Elsevier Inc.保留所有权利。
It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of similar to 10 angstrom, but maps with resolutions of 5 angstrom or better are still celebrated events. The electron microscope has a resolving power to better than 2 angstrom, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4 angstrom (FSC0.5) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined. (C) 2008 Elsevier Inc. All rights reserved.