高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (11): 2335.doi: 10.7503/cjcu20200323
• 庆祝《高等学校化学学报》复刊40周年专栏 • 上一篇 下一篇
赵关芳1,2, 邹天一1,2, 程思航1,2, 于洋1,2, 王慧利1, 王宏达1,2()
收稿日期:
2020-06-04
出版日期:
2020-11-10
发布日期:
2020-11-06
通讯作者:
王宏达
E-mail:hdwang@ciac.ac.cn
基金资助:
ZHAO Guanfang1,2, ZOU Tianyi1,2, CHENG Sihang1,2, YU Yang1,2, WANG Huili1, WANG Hongda1,2()
Received:
2020-06-04
Online:
2020-11-10
Published:
2020-11-06
Contact:
WANG Hongda
E-mail:hdwang@ciac.ac.cn
Supported by:
摘要:
对生物大分子复合物的研究和结构分析对于全面了解其功能和生物学意义至关重要. 冷冻电子显微镜在提供生物大分子结构及大分子分布等方面起到重要的作用. 近年来, 冷冻电子显微镜的硬件和软件的发展进一步提高了冷冻电子显微镜的有效性, 使其对各种生物结构、 蛋白质结构的解析更加准确快捷. 但是, 对于生物系统来说, 蛋白质和大分子复合物等均处于复杂的生理环境中, 因此原位检测生物分子的三维结构对于生物体系和结构生物学具有重要意义. 冷冻电子断层扫描作为一种功能强大的技术, 可以无需标记直接通过冷冻样品的固有衬度识别生物大分子的结构, 并且可在原位生理环境中对生物分子进行纳米级分辨率的三维成像. 本文综述了与冷冻电子断层扫描相关的样品制备和数据处理技术, 并总结了冷冻电子断层扫描技术在分离的大分子复合物和整个细胞或组织中的生物学应用.
中图分类号:
TrendMD:
赵关芳, 邹天一, 程思航, 于洋, 王慧利, 王宏达. 原位冷冻电子断层扫描的发展及在生物研究方面的应用. 高等学校化学学报, 2020, 41(11): 2335.
ZHAO Guanfang, ZOU Tianyi, CHENG Sihang, YU Yang, WANG Huili, WANG Hongda. Developments of in situ Cryo-electron Tomography for Biological Applications. Chem. J. Chinese Universities, 2020, 41(11): 2335.
Fig.1 Methods of preparing samples suitable for cryo?ET(A) Samples were prepared by plunge freezing[32]: sample solution is added to the carbon support films and using filter paper to remove excess solution from one or both sides; grid is rapidly plunged into cryogen precooled at liquid nitrogen temperature and imaged with cryo?ET; (B) schematic diagram of high pressure freezing[48]; (C) tomographic slice of a mitotic Hela S3 cell and the oblique striations of the image intensity is the knife marks[39]; (D) vitrified sample is micromachined using FIB milling and the patterns(red boxes) are chosen depending on the milling geometry[49].(A) Copyright 2017, Elsevier; (B) Copyright 2014, Springer Science Business Media; (C) Copyright 2005, Elsevier; (D) Copyright 2013, Elsevier.
Fig.2 Principles of data collection and processing in cryo?ET[14,56](A) Schematic diagram of cryo?EM; (B) samples embedded in ice is tilted by a certain range of angles and record the projec?ted images at each angle; (C) the tilt series of projected images collected around a common axis; (D) the projected images in the tilt series are aligned with their common axis through calculation and reconstructed into a 3D tomogram by weighted projection or other method; (E) sub?tomograms representing 3D view of single macromolecules can be extracted from the reconstructed tomogram, and then aligned and averaged. (A) Copyright 2015, Elsevier; (B—E) Copyright 2017, Springer Nature.
Fig.3 Procedures to identify structures of interest in tomograms(A) Schematic flow diagram of template matching showing the detection and identification strategy[62]; (B) training and automated annotation process of convolutional neural networks for identifying features of interest in tomograms[65]; (C) tracing density at a synapes from rodent cerebrocortical synaptosomal preparation through Pyseg without templates[68]: tomographic slice(left), Morce complex(right).(A) Copyright 2002, The National Academy of Sciences; (B) Copyright 2017, Springer Nature; (C) Copyright 2020, Springer Nature.
Fig.4 Some biological applications of cryo?ET(A) Tomographic slices of polysome organizations(left) and the corresponding isosurface of single ribosomes(right)[78]; (B) immature HIV virus: tomographic slices(left) and fourier shell correlation plot before (red) and after(green) CTF correction(right)[80]; (C) identifyexcitatory and inhibitory synapses by combining cryo?ET and fluorescence microscope. Tomographic slices of excita?tory(green fluorescence) and inhibitory(red fluorescence) synapses(left). Zoom?in views of the boxed area(right) show the differnce between excitatory and inhibitory synapses(red dashed lines)[84]; (D) template matching in L. interrogans cells[88](top: template library of proteins; middle: representative subvolume of cells; bottom: corresponding surface?rendered models).(A) Copyright 2009, Elsevier; (B) Copyright 2009, National Academy of Sciences; (C) Copyright 2018, Society for Neuroscience; (D) Copyright 2009, Springer Nature.
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