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化学进展 2013, Vol. 25 Issue (05): 669-676 DOI: 10.7536/PC121105 前一篇   后一篇

• 综述与评论 •

IPET:测定独个生物大分子三维空间结构的实验方法

张腾1,2, 彭云辉1,2, 童慧敏1,2, Matthew J Rames1, 张磊1, 任罡*1   

  1. 1. The Molecular Foundry,Lawrence Berkeley National Laboratory,Berkeley,CA 94720, USA;
    2. 西安交通大学理学院 西安 710049
  • 收稿日期:2012-11-01 修回日期:2012-12-01 出版日期:2013-05-24 发布日期:2013-04-15
  • 通讯作者: 任罡 E-mail:gren@lbl.gov
  • 基金资助:

    美国能源部基础科学基金项目(No.DE-AC02-05CH11231)和美国国立卫生研究院(NIH)基金项目(No.R01HL115153,R01GM1044227)资助

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IPET: An Experimental Method to Determine the 3-Dimensional Structure of An Individual Macromolecule

Zhang Teng1,2, Peng Yunhui1,2, Tong Huimin1,2, Matthew J Rames1, Zhang Lei1, Ren Gang*1   

  1. 1. The Molecular Foundry,Lawrence Berkeley National Laboratory,Berkeley,CA 94720, USA;
    2. School of Science, Xi’an Jiaotong University, Xi’an 710049, China
  • Received:2012-11-01 Revised:2012-12-01 Online:2013-05-24 Published:2013-04-15

蛋白质的动态特性和结构活性对于蛋白质功能的调控具有根本意义。传统的结构确定方法(包括X射线和电子显微镜单颗粒分析技术等)往往需要成千上万不同蛋白质分子的平均信号,因此难以确定蛋白质分子的动态结构。而电子显微断层成像技术是一种对独个生物个体结构从不同的观测角度照相、并计算来恢复该个体的三维结构密度图的方法。传统的冷冻电子断层成像重构方法采用整个大尺寸电镜图像进行重构计算,通常用来研究细菌、细胞切片等大尺寸生物个体在较低分辨率下的结构;由于分辨率的限制,不足以获得小尺寸的蛋白质分子的结构细节。最近,任罡研究小组提出一种独个生物颗粒的电子显微断层成像方法(individual-particle electron tomography,IPET)。 该方法通过减小图像尺寸(直至所选区域只包含单个蛋白质分子)的策略,运用提出的FETR(focused electron tomography reconstruction)算法来提高独个大分子重构的分辨率。 此方法不需要初始模型和大量分子的平均信号,同时能够容忍一定的测角误差。 本文综述了IPET/FETR方法在确定独个分子结构过程中的具体步骤以及如何应用该方法来研究蛋白动态特性和结构变化特征。 期望通过该综述和国内同行交流,分享最新的前沿研究,为赶超世界科技前沿的建设添砖加瓦。

关键词： 蛋白质动态结构, 独个蛋白质结构, 单分子电子断层成像方法, FETR算法

Dynamic personalities and structural heterogeneities of proteins are essential for understanding their proper functions. However,structure determination of dynamic/heterogeneous protein is limited by current technologies, such as X-ray crystallography and electron microscopy (EM) single particle analysis, both of which generally require averaging from thousands of different proteins based on an assumption that these thousand proteins are structurally identical. Electron tomography (ET) provides a tool for visualization of a unique biological object from a series of tilted viewing angles. Conventional reconstruction methods using whole micrographs provide tools for 3-dimensional (3D) reconstructions of a large biological object, such as bacteria, and sections of cell. However, for small and low-symmetry proteins, these methods have limited power in reconstruction resolution. Recently, Ren’s group reported a so-called individual-particle electron tomography (IPET) method, in which, a “focused electron tomography reconstruction (FETR)” algorithm was proposed to improve the reconstruction resolution by decreasing the image size so that it only contains a single-instance protein. IPET method requires no pre-given initial model or average of multiple molecules, but also can tolerate certain levels of measuring tilt errors. In this review, we demonstrate the IPET/FETR method in detail to share this current progress with the researchers in China. We believed IPET is a new and robust approach to determine the structure of a single/individual molecule that is a basis for studying the dynamic character and structural heterogeneity of macromolecule via comparison and structural analyses of structures determined from different individual macromolecules. Contents
1 Introduction
2 IPET method and FETR algorithm
2.1 Generating simulated cryoET data
2.2 Basic tools for image processing
2.3 Focused electron tomography reconstruction (FETR) algorithm
3 Validation of focused ET reconstruction method by real experimental data
3.1 3D reconstruction of a human IgG antibody by negative-staining ET
3.2 3D reconstruction of a high-density lipoprotein by cryoET
4 Conclusion and outlook

Key words: protein dynamics, an individual protein structure, individual-particle electron tomography (IPET), focused electron tomography reconstruction (FETR) algorithm

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