Journal of Shanghai Jiao Tong University (Medical Science) ›› 2022, Vol. 42 ›› Issue (4): 455-463.doi: 10.3969/j.issn.1674-8115.2022.04.007

• Basic research • Previous Articles     Next Articles

Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex

DUAN Yujuan(), HUANG Jing()   

  1. Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
  • Received:2022-01-13 Accepted:2022-03-07 Online:2022-04-28 Published:2022-04-28
  • Contact: HUANG Jing;
  • Supported by:
    National Natural Science Foundation of China(32022036)

Abstract: Objective

·To study the structure of human nucleosome remodeling and deacetylase complex (NuRD complex) with negative-stain electron microscopy to obtain the profile information of human NuRD complex.


·Full length MBD3 (methyl-CpG binding domain protein 3) and GATAD2A (GATA zinc finger domain containing 2A) constructs were cloned into the pMLink vectors with an amino terminal 10×His tag and a carboxyl terminal 3×Flag affinity tag, respectively. Proteins were expressed in human Expi293F cells grown in suspension cultures by using polyethylenimine as the transfection reagent, and were isolated via affinity purifications with Ni-NTA and anti-DYKDDDDK (Flag) G1 affinity resin sequentially. The complex was further purified by Superose 6 Increase 5/150 gel filtration chromatography to improve the homogeneity, identified by Western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and was then studied by negative-stain electron microscopy and single particle analysis. The existing atomic structural model of the subcomplex (7AO9, 5FXY) in Protein Data Bank (PDB) was automatically docked into the generated structural model by the UCSF Chimera software to predict the localization of multiple protein components in the structural model.


·The Flag-tagged MBD3 and His-tagged GATAD2A proteins could effectively pull down the other endogenous components of the NuRD complex through two-step affinity purifications. The high-purity complex was obtained by gel filtration chromatography and confirmed as the NuRD complex by LC-MS/MS and Western blotting identification. Preliminary study on the three-dimensional (3D) structure of the NuRD complex was carried out with negative-stain electron microscopy and single particle analysis, which revealed that the NuRD complex was in the obvious shape of a long asymmetrical rod. The 3D structural model of the human NuRD complex was finally obtained by further 3D refinement at an overall resolution of 17 ? (1 ?=0.1 nm). The existing atomic structural model (PDB: 7AO9, 5FXY) was automatically docked into the negative staining structural model, and the localizations of MTA1/2/3 (metastasis-associated protein 1/2/3), HDAC1/2 (histone deacetylase 1/2), RBBP4/7 (retinoblastoma-binding protein 4/7) and MBD2/3 (methyl-CpG-binding domain protein 2/3) subunits in the NuRD complex were preliminarily confirmed.


·A low-resolution negative-staining structural model of human NuRD complex is obtained by single particle analysis.

Key words: nucleosome remodeling and deacetylase (NuRD) complex, epigenetic regulation, deacetylation, negative-stain electron microscopy

CLC Number: