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Open data
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Basic information
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Title | CryoEM structure of wild-type GAPDH | |||||||||
![]() | GAPDH, 0h Oxidation, WT | |||||||||
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![]() | GAPDH / energy / cytosolic protein / TRANSFERASE | |||||||||
Function / homology | ![]() 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 / glucose metabolic process / NAD binding / microtubule cytoskeleton / disordered domain specific binding / antimicrobial humoral immune response mediated by antimicrobial peptide / 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 / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 1.98 Å | |||||||||
![]() | Choi WY / Wu H / Cheng YF / Manglik A | |||||||||
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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 49.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21.8 KB 21.8 KB | Display Display | ![]() |
Images | ![]() | 131 KB | ||
Filedesc metadata | ![]() | 5.3 KB | ||
Others | ![]() ![]() ![]() ![]() | 1.4 MB 1.4 MB 39.7 MB 39.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8g17MC ![]() 8g12C ![]() 8g13C ![]() 8g14C ![]() 8g15C ![]() 8g16C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | GAPDH, 0h Oxidation, WT | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.835 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: GAPDH, 0h Oxidation, WT, single subunit analysis, C1, active subunit
File | emd_29664_additional_3.map | ||||||||||||
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Annotation | GAPDH, 0h Oxidation, WT, single subunit analysis, C1, active subunit | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: GAPDH, 0h Oxidation, WT, single subunit analysis, C1, active subunit
File | emd_29664_additional_4.map | ||||||||||||
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Annotation | GAPDH, 0h Oxidation, WT, single subunit analysis, C1, active subunit | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: GAPDH, 0h Oxidation, WT, halfmap2
File | emd_29664_half_map_1.map | ||||||||||||
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Annotation | GAPDH, 0h Oxidation, WT, halfmap2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: GAPDH, 0h Oxidation, WT, halfmap1
File | emd_29664_half_map_2.map | ||||||||||||
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Annotation | GAPDH, 0h Oxidation, WT, halfmap1 | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : GAPDH
Entire | Name: GAPDH |
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Components |
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-Supramolecule #1: GAPDH
Supramolecule | Name: GAPDH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: Glyceraldehyde-3-phosphate dehydrogenase
Macromolecule | Name: Glyceraldehyde-3-phosphate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO EC number: glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 35.967969 KDa |
Sequence | String: GKVKVGVNGF GRIGRLVTRA AFNSGKVDIV AINDPFIDLN YMVYMFQYDS THGKFHGTVK AENGKLVING NPITIFQERD PSKIKWGDA GAEYVVESTG VFTTMEKAGA HLQGGAKRVI ISAPSADAPM FVMGVNHEKY DNSLKIISNA SCTTNCLAPL A KVIHDNFG ...String: GKVKVGVNGF GRIGRLVTRA AFNSGKVDIV AINDPFIDLN YMVYMFQYDS THGKFHGTVK AENGKLVING NPITIFQERD PSKIKWGDA GAEYVVESTG VFTTMEKAGA HLQGGAKRVI ISAPSADAPM FVMGVNHEKY DNSLKIISNA SCTTNCLAPL A KVIHDNFG IVEGLMTTVH AITATQKTVD GPSGKLWRDG RGALQNIIPA STGAAKAVGK VIPELNGKLT GMAFRVPTAN VS VVDLTCR LEKPAKYDDI KKVVKQASEG PLKGILGYTE HQVVSSDFNS DTHSSTFDAG AGIALNDHFV KLISWYDNEF GYS NRVVDL MAHMASKE UniProtKB: Glyceraldehyde-3-phosphate dehydrogenase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 8 |
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Vitrification | Cryogen name: OTHER |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.8 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: OTHER |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 1.98 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 373104 |
Initial angle assignment | Type: ANGULAR RECONSTITUTION |
Final angle assignment | Type: ANGULAR RECONSTITUTION |