Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.
Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.
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DOI：
10.1016/j.ymeth.2016.03.007
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发表时间：
2016-05-01
期刊：
影响因子：
--
通讯作者：
Topf M
中科院分区：
文献类型：
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作者：
Joseph AP;Malhotra S;Burnley T;Wood C;Clare DK;Winn M;Topf M
随着生物大分子三维电子显微镜重建分辨率的不断提高，需要更高分辨率的拟合和细化方法以及更可靠的模型评估方法。Flex-EM/ modeler已被用于不同生物系统的中低分辨率密度图中原子模型的柔性拟合。在这里，我们使用模拟和实验数据，包括新处理的Apo-GroEL地图，证明了该方法在更高分辨率（2.5-4.5 Å）下成功改进结构的适用性。采用了一种层次细化协议，其中刚体定义放宽，原子位移步骤在细化的连续阶段逐步减少。对于局部拟合的评估，我们使用SMOC（基于片段的Manders '重叠系数）评分，而使用Qmean评分来检查模型质量。比较不同精化阶段的SMOC剖面有助于检测拟合不良的区域。我们还展示了初始模型误差如何对拟合优度产生重大影响。最后，我们讨论了Flex-EM在CCP-EM软件套件中的实现。
As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5–4.5 Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders’ overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.