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- PDB-8g14: CryoEM structure of cytosolic GAPDH under 8h Oxidative Stress, class2 -
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Open data
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Basic information
Entry | Database: PDB / ID: 8g14 | ||||||
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Title | CryoEM structure of cytosolic GAPDH under 8h Oxidative Stress, class2 | ||||||
![]() | Glyceraldehyde-3-phosphate dehydrogenase![]() | ||||||
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Function / homology | ![]() peptidyl-cysteine S-trans-nitrosylation / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Choi, W.Y. / Wu, H. / Cheng, Y.F. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM. Authors: Wooyoung Choi / Hao Wu / Klaus Yserentant / Bo Huang / Yifan Cheng / ![]() Abstract: CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor ...CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 248.5 KB | Display | ![]() |
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PDB format | ![]() | 201.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 29661MC ![]() 8g12C ![]() 8g13C ![]() 8g15C ![]() 8g16C ![]() 8g17C M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | ![]() Mass: 35967.969 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() References: UniProt: P04406, ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: CELL / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: GAPDH![]() |
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Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() |
Vitrification![]() | Cryogen name: OTHER |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() |
Image recording | Electron dose: 45.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction![]() | Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 337177 / Symmetry type: POINT | ||||||||||||||||||||||||
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