A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion
A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion
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DOI：
10.3791/63387
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发表时间：
2022-01-01
影响因子：
1.2
通讯作者：
Kulczyk, Arkadiusz W.
中科院分区：
文献类型：
--
作者：
DiIorio, Megan C.;Kulczyk, Arkadiusz W.
仪器和图像处理软件的最新进展使单粒子冷冻电子显微镜（cryo-EM）成为结构生物学家确定各种大分子高分辨率结构的首选方法。多个软件套件可供新用户和专家用户用于图像处理和结构计算，简化了相同的基本工作流程：显微镜检测器采集的电影进行光束诱导运动和对比度传递函数（CTF）估计的校正。接下来，从平均电影帧中选择并提取粒子图像，用于迭代2D和3D分类，然后进行3D重建、细化和验证。由于各种软件包采用不同的算法，并需要不同水平的专业知识来操作，因此它们生成的3D地图通常在质量和分辨率上有所不同。因此，用户定期在各种程序之间传输数据，以获得最佳结果。本文为用户提供了在流行软件包（cryoSPARC v3、RELION-3和Scipion 3）中导航工作流程的指南，以获得腺相关病毒（AV）的近原子分辨率结构。我们首先详细介绍了cryoprotein v3的图像处理管道，因为其高效的算法和易于使用的GUI允许用户快速获得3D地图。在下一步中，我们使用PyEM和内部脚本将粒子坐标从cryoprotein v3中获得的最佳质量3D重建转换和传输到RELION-3和Scipion 3，并重新计算3D地图。最后，我们概述了通过集成RELION-3和Scipion 3的算法进一步改进和验证所得结构的步骤。在这篇文章中，我们描述了如何有效地利用三个处理平台来创建一个单一的和强大的工作流程，适用于各种数据集的高分辨率结构测定。
Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adenoassociated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.