Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis | |
Wang, Huping1,2; Han, Wenyu1,2; Cong, Yao1,2; Takagi, Junichi3 | |
刊名 | JOURNAL OF MOLECULAR BIOLOGY |
2018 | |
卷号 | 430期号:10页码:1417-1425 |
关键词 | Particle Electron-microscopy Eukaryotic Chaperonin Cryoelectron Microscopy Molecular Chaperones Crystal-structure Pa Tag Protein Cct Resolution Tric/cct |
ISSN号 | 0022-2836 |
DOI | 10.1016/j.jmb.2018.03.026 |
文献子类 | Article |
英文摘要 | Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. (C) 2018 Elsevier Ltd. All rights reserved. |
电子版国际标准刊号 | 1089-8638 |
WOS研究方向 | Biochemistry & Molecular Biology |
语种 | 英语 |
WOS记录号 | WOS:000433642500002 |
内容类型 | 期刊论文 |
版本 | 出版稿 |
源URL | [http://202.127.25.143/handle/331003/3513] |
专题 | 生化所2018年发文 上海生化细胞研究所_上海生科院生化细胞研究所 |
通讯作者 | Cong, Yao |
作者单位 | 1.Univ Chinese Acad Sci, CAS Ctr Excellence Mol Cell Sci, Natl Ctr Prot Sci Shanghai,State Key Lab Mol Biol, Chinese Acad Sci,Shanghai Inst Biochem & Cell Bio, Shanghai, Peoples R China; 2.Chinese Acad Sci, Shanghai Sci Res Ctr, Shanghai, Peoples R China; 3.Osaka Univ, Inst Prot Res, Lab Prot Synth & Express, Osaka, Japan |
推荐引用方式 GB/T 7714 | Wang, Huping,Han, Wenyu,Cong, Yao,et al. Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis[J]. JOURNAL OF MOLECULAR BIOLOGY,2018,430(10):1417-1425. |
APA | Wang, Huping,Han, Wenyu,Cong, Yao,&Takagi, Junichi.(2018).Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis.JOURNAL OF MOLECULAR BIOLOGY,430(10),1417-1425. |
MLA | Wang, Huping,et al."Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis".JOURNAL OF MOLECULAR BIOLOGY 430.10(2018):1417-1425. |
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