冷冻电镜解析病毒颗粒中的蛋白质结构

朱东杰,章新政*

冷冻电镜解析病毒颗粒中的蛋白质结构

朱东杰,章新政*

(1.中国科学院生物物理研究所生物大分子国家重点实验室,北京100101;

2.中国科学院大学,北京 100049)

摘   要  病毒的蛋白质衣壳在病毒颗粒识别受体以及入侵细胞中起到了关键作用,其高分辨结构的解析有利于帮助理解病毒入侵宿主的分子机制,并为对抗病毒的疫苗和药物的设计提供精细的靶点信息。由于正二十面体病毒粒子的高对称性以及其在冷冻电镜照片中远优于蛋白质复合物的衬度,伴随着技术发展,冷冻电镜单颗粒技术率先在一系列直径约70 nm的正二十面体病毒上获得了准原子分辨率结构解析,这些工作不仅揭示了病毒颗粒精细的组装机制,也展示了冷冻电镜解析蛋白质结构的潜力。随着直接电子探测相机的出现,更小的正二十面体以及其它对称性病毒的三维重构分辨率获得显著提升。而埃瓦尔德球矫正算法的发展以及其在病毒重构中的应用使大型正二十面体病毒三维重构突破了分辨率瓶颈,最终实现了准原子分辨率结构解析,在原子尺度展示了极其复杂的大型病毒是如何由成百上千个结构蛋白组装而成。现阶段,冷冻电镜技术正在向原位结构解析方向发展。原位结构解析技术不仅可用于非对称性病毒颗粒上蛋白质的高分辨结构解析,也可以用于研究各种病毒入侵宿主细胞、转录、复制以及病毒颗粒组装、出芽的研究。基于电子断层重构以及亚单位平均的原位结构解析方法展示了其强大的能力,但依然存在着数据采集通量低,三维重构分辨率提升困难的问题。原位结构解析需要进一步发展、完善,结合更好的细胞冷冻切片制备技术,将病毒的原位结构研究推进到新的高度。本文将结合冷冻电镜重构技术的发展和病毒粒子的结构生物学研究,描述这三个阶段的技术和科研的进展,并对未来进行展望。

关键词   冷冻电镜;病毒结构解析;正二十面体;对称性破缺;原位结构解析

中图分类号:Q6-33;Q937   文献标识码:A

 

Protein structures on virus particles resolved by Cryo-EM

ZHU Dong-jie 1 ,ZHANG Xin-zheng 12*

(1.National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100100;

2. University of Chinese Academy of Sciences, Beijing100049,China)

 

Abstract   The structure protein of virus plays a key role in recognition and invasion of host cells. High-resolution models of such proteins help understanding their molecular mechanism in atomic level, and provide detailed information of target for vaccine and pharmaceutical design. Since the high-symmetry and higher contrast of icosahedral viruses in Cryo-electron microscopy (Cryo-EM) images than general protein complexes, icosahedral viruses with diameter being around 70 nm became the first sample to achieve near-atomic resolution by single particle method in Cryo-EM. Such beautiful works not only presented the tidy assembly mechanism, but also showed the great potential of Cryo-EM in structural biology. As the development and application of direct electron detector in Cryo-EM, smaller viruses with icosahedral or other symmetry were resolved in significant higher resolution. Algorithms correcting the Ewald’s sphere effect broke the resolution limitation to viruses and near-atomic resolution could be achieved on viruses larger than 70 nm. These works showed how such complex virus was assembled accurately by thousands of proteins. Nowadays, in situ reconstruction in Cryo-EM is in fast growth. In situ reconstruction can either be used in high-resolution reconstructions of asymmetric viruses, or reveal how viruses enter the host cell, assemble inside cell and being released from the cells. Cryo-electron tomography and sub-tomogram averaging have already showed their great potential in in situ reconstruction. However, the low throughput of data acquisition, the toughness to push the resolution of reconstruction are still major issues during applications. Combining with finer cryogenic slicing technology, in situ reconstruction could push the structural study of viruses to a new edge with further development. In this review, we describe the technique developments in Cryo-EM and the typical achievements on the structural study of viruses and discuss its possible future. 

Keywords  Cryo-EM;virus structure;icosahedral;symmetry relax;in situreconstruction

 

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