Single particle cryo-electron microscopy and 3-D reconstruction of viruses.
Single particle cryo-electron microscopy and 3-D reconstruction of viruses.
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DOI：
10.1007/978-1-62703-776-1_19
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Jiang, Wen
中科院分区：
文献类型：
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作者：
Guo, Fei;Jiang, Wen
随着仪器、图像处理算法和计算资源的快速进步，二十面体病毒的单粒子电子冷冻显微镜 (cryo-EM) 3D 重建现已达到接近原子分辨率 (3-4 Å)。凭借可比的分辨率和更可预测的结果，冷冻电镜现在被认为是优于 X 射线晶体学的确定二十面体病毒原子结构的首选方法。在近原子分辨率下，可以可靠地构建全原子模型或主干模型，从而可以在残留水平上了解病毒组装和病毒生命周期不同阶段之间的构象变化。随着不对称重建的发展，现在可以可视化复杂病毒的完整结构，不仅具有二十面体外壳，而且还具有多个非二十面体结构特征。在本章中，我们将描述单粒子冷冻电镜实验和计算程序，用于二十面体病毒的近原子分辨率重建和具有二十面体和非二十面体结构成分的病毒的不对称重建。还介绍了使用真正独立的从头初始模型和改进对重建和分辨率评估进行严格验证的程序。
With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.