+Open data
-Basic information
Entry | Database: PDB / ID: 8g12 | ||||||
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Title | CryoEM structure of nuclear GAPDH under 8h Oxidative Stress | ||||||
Components | Glyceraldehyde-3-phosphate dehydrogenase | ||||||
Keywords | TRANSFERASE / GAPDH / energy / cytosolic protein | ||||||
Function / homology | Function and homology information peptidyl-cysteine S-trans-nitrosylation / Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / peptidyl-cysteine S-nitrosylase activity / killing by host of symbiont cells / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / aspartic-type endopeptidase inhibitor activity / negative regulation of endopeptidase activity / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / Gluconeogenesis / GAIT complex ...peptidyl-cysteine S-trans-nitrosylation / Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / peptidyl-cysteine S-nitrosylase activity / killing by host of symbiont cells / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / aspartic-type endopeptidase inhibitor activity / negative regulation of endopeptidase activity / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / Gluconeogenesis / GAIT complex / Glycolysis / positive regulation of type I interferon production / regulation of macroautophagy / defense response to fungus / lipid droplet / positive regulation of cytokine production / glycolytic process / cellular response to type II interferon / microtubule cytoskeleton organization / antimicrobial humoral immune response mediated by antimicrobial peptide / glucose metabolic process / microtubule cytoskeleton / NAD binding / disordered domain specific binding / NADP binding / microtubule binding / nuclear membrane / neuron apoptotic process / positive regulation of canonical NF-kappaB signal transduction / vesicle / killing of cells of another organism / protein stabilization / negative regulation of translation / ribonucleoprotein complex / intracellular membrane-bounded organelle / perinuclear region of cytoplasm / extracellular exosome / identical protein binding / membrane / nucleus / plasma membrane / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.17 Å | ||||||
Authors | Choi, W.Y. / Wu, H. / Cheng, Y.F. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2023 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: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g12.cif.gz | 249 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g12.ent.gz | 201.2 KB | Display | PDB format |
PDBx/mmJSON format | 8g12.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8g12_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8g12_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8g12_validation.xml.gz | 42.6 KB | Display | |
Data in CIF | 8g12_validation.cif.gz | 59.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g1/8g12 ftp://data.pdbj.org/pub/pdb/validation_reports/g1/8g12 | HTTPS FTP |
-Related structure data
Related structure data | 29659MC 8g13C 8g14C 8g15C 8g16C 8g17C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 35983.969 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: GAPDH, GAPD, CDABP0047, OK/SW-cl.12 / Production host: Homo (humans) References: UniProt: P04406, glyceraldehyde-3-phosphate dehydrogenase (phosphorylating), Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: CELL / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: GAPDH / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: Homo (humans) |
Source (recombinant) | Organism: Homo sapiens (human) |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: OTHER |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 45.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 764164 / Symmetry type: POINT |