Journal of Shanghai Jiao Tong University (Medical Science) >

2022 , Vol. 42 >Issue 3: 290 - 297

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.03.005

Innovative research team achievement column

Electron microscopic study of Shelterin quinary complex structure in fission yeast

  • Yanjia LU ,
  • Hong SUN ,
  • Zhenfang WU ,
  • Ming LEI
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  • Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
LEI Ming, E-mail: leim@shsmu.edu.cn.

Received date: 2021-12-27

  Online published: 2022-05-09

Supported by

National Key R&D Program of China(2018YFA0107004);National Natural Science Foundation of China(31930063)

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Abstract

Objective

·To study the structure of the Shelterin quinary complex (Rap1-Poz1-Tpz1-Ccq1-Pot1) in fission yeast Schizosaccharomyces pombe (S. pombe)by negative-staining and electron microscopy.

Methods

·The Shelterin quinary complex was reconstituted via expression and purification of recombinant proteins in Escherichia coli with histidine-small ubiquitin-related modifier (His-SUMO) and glutathione S-transferase (GST) tags. The target protein complex was isolated via sequential Ni-NTA and glutathione sepharose affinity purification followed by gel filtration chromatography (SuperoseTM6 10/300 GL). The protein samples were stained by 0.75% uranium formate, and then observed by 120 kV transmission electron microscope (TEM) with a magnification of 92 000. The micrographs with good particle dispersity were obtained and the 3D structure of Shelterin complex was reconstructed by single particle image analysis by using EMAN2 and Relion3.0. Finally, UCSF Chimera was used to analyze the reconstruction model of Shelterin complex.

Results

·The recombinant S. pombe Shelterin quinary complex with high purity, component integrity and good homogeneity was obtained by using a tandem affinity purification scheme with Ni-NTA and GST-based chromatography. Eighty-three micrographs were collected through 120 kV TEM, in which 18 659 particles were picked for 3D model reconstruction. After that, the rough 3D structure of the complex was obtained. Molecular docking of the available crystal structures of Shelterin subcomplex into the reconstructed model revealed that the complex existed in a dimeric form and determined the positioning of each component in the complex.

Conclusion

·The low-resolution 3D model of S. pombe Shelterin quinary complex (Rap1-Poz1-Tpz1-Ccq1-Pot1) is obtained.

Key words： telomere; telomere end-protection; Shelterin complex; electron microscopy; negative-staining

Cite this article

Yanjia LU , Hong SUN , Zhenfang WU , Ming LEI . Electron microscopic study of Shelterin quinary complex structure in fission yeast[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022 , 42(3) : 290 -297 . DOI: 10.3969/j.issn.1674-8115.2022.03.005

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