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Open data
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Basic information
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Title | CryoEM structure of nuclear GAPDH under 8h Oxidative Stress | |||||||||
![]() | GAPDH,Nuclear Oxidation 8h,D2 | |||||||||
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Function / homology | ![]() peptidyl-cysteine S-trans-nitrosylation / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | 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 | ![]() | 39.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 24.1 KB 24.1 KB | Display Display | ![]() |
Images | ![]() | 61.4 KB | ||
Filedesc metadata | ![]() | 5.5 KB | ||
Others | ![]() ![]() ![]() ![]() ![]() ![]() ![]() | 1.3 MB 1.3 MB 1.3 MB 1.3 MB 1.3 MB 39.7 MB 39.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8g12MC ![]() 8g13C ![]() 8g14C ![]() 8g15C ![]() 8g16C ![]() 8g17C 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,Nuclear Oxidation 8h,D2 | ||||||||||||||||||||
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,Nuclear Oxidation 8h,single subunit analysis,inactive
File | emd_29659_additional_1.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive
File | emd_29659_additional_2.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
File | emd_29659_additional_3.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
File | emd_29659_additional_4.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
File | emd_29659_additional_5.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: GAPDH,Nuclear Oxidation 8h,D2,halfmap1
File | emd_29659_half_map_1.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,D2,halfmap1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: GAPDH,Nuclear Oxidation 8h,D2,halfmap2
File | emd_29659_half_map_2.map | ||||||||||||
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Annotation | GAPDH,Nuclear Oxidation 8h,D2,halfmap2 | ||||||||||||
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: ![]() |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 35.983969 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: GKVKVGVNGF GRIGRLVTRA AFNSGKVDIV AINDPFIDLN YMVYMFQYDS THGKFHGTVK AENGKLVING NPITIFQERD PSKIKWGDA GAEYVVESTG VFTTMEKAGA HLQGGAKRVI ISAPSADAPM FVMGVNHEKY DNSLKIISNA S(CSO)TTNCL AP LAKVIHDNFG ...String: GKVKVGVNGF GRIGRLVTRA AFNSGKVDIV AINDPFIDLN YMVYMFQYDS THGKFHGTVK AENGKLVING NPITIFQERD PSKIKWGDA GAEYVVESTG VFTTMEKAGA HLQGGAKRVI ISAPSADAPM FVMGVNHEKY DNSLKIISNA S(CSO)TTNCL AP LAKVIHDNFG IVEGLMTTVH AITATQKTVD GPSGKLWRDG RGALQNIIPA STGAAKAVGK VIPELNGKLT GMAFRVPT A NVSVVDLTCR LEKPAKYDDI KKVVKQASEG PLKGILGYTE HQVVSSDFNS DTHSSTFDAG AGIALNDHFV KLISWYDNE FGYSNRVVDL MAHMASKE UniProtKB: ![]() |
-Experimental details
-Structure determination
Method | ![]() |
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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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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD![]() |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.8 e/Å2 |
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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Initial angle assignment | Type: ANGULAR RECONSTITUTION |
Final angle assignment | Type: ANGULAR RECONSTITUTION |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 764164 |