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• W2796192703 startingPage "1417" @default.
• W2796192703 abstract "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." @default.
• W2796192703 created "2018-04-13" @default.
• W2796192703 creator A5016321178 @default.
• W2796192703 creator A5026230484 @default.
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• W2796192703 date "2018-05-01" @default.
• W2796192703 modified "2023-09-24" @default.
• W2796192703 title "Yeast Inner-Subunit PA–NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis" @default.
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• W2796192703 doi "https://doi.org/10.1016/j.jmb.2018.03.026" @default.
• W2796192703 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29625202" @default.
• W2796192703 hasPublicationYear "2018" @default.
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