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- EMDB-29663: CryoEM structure of cytoplasmic GAPDH under 24h Oxidative Stress -

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Basic information

Entry
Database: EMDB / ID: EMD-29663
TitleCryoEM structure of cytoplasmic GAPDH under 24h Oxidative Stress
Map dataGAPDH, cyto, oxidative stress 24h, overall, d2Glyceraldehyde 3-phosphate dehydrogenase
Sample
  • Complex: GAPDHGlyceraldehyde 3-phosphate dehydrogenase
    • Protein or peptide: Glyceraldehyde-3-phosphate dehydrogenaseGlyceraldehyde 3-phosphate dehydrogenase
KeywordsGAPDH / energy / cytosolic protein / TRANSFERASE
Function / homology
Function and homology information


Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / peptidyl-cysteine S-nitrosylase activity / peptidyl-cysteine S-trans-nitrosylation / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / killing by host of symbiont cells / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / negative regulation of endopeptidase activity / aspartic-type endopeptidase inhibitor activity / Gluconeogenesis / GAIT complex ...Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / peptidyl-cysteine S-nitrosylase activity / peptidyl-cysteine S-trans-nitrosylation / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / killing by host of symbiont cells / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / negative regulation of endopeptidase activity / aspartic-type endopeptidase inhibitor 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 / microtubule cytoskeleton organization / cellular response to type II interferon / glucose metabolic process / NAD binding / microtubule cytoskeleton / disordered domain specific binding / antimicrobial humoral immune response mediated by antimicrobial peptide / NADP binding / microtubule binding / neuron apoptotic process / positive regulation of canonical NF-kappaB signal transduction / nuclear membrane / killing of cells of another organism / vesicle / negative regulation of translation / protein stabilization / ribonucleoprotein complex / intracellular membrane-bounded organelle / perinuclear region of cytoplasm / extracellular exosome / membrane / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Glyceraldehyde-3-phosphate dehydrogenase, type I / Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD(P) binding domain / Glyceraldehyde 3-phosphate dehydrogenase, catalytic domain / Glyceraldehyde/Erythrose phosphate dehydrogenase family / Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain / Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Glyceraldehyde-3-phosphate dehydrogenase
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.07 Å
AuthorsChoi WY / Wu H / Cheng YF / Manglik A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD) United States
CitationJournal: 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
DepositionFeb 1, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29663.png

Downloads & links

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Map

FileDownload / File: emd_29663.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGAPDH, cyto, oxidative stress 24h, overall, d2
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.0073
Minimum - Maximum-0.01112765 - 0.030894276
Average (Standard dev.)0.000051653245 (±0.0011373608)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 200.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: GAPDH, cyto, oxidative stress 24h, active, single subunit...

Fileemd_29663_additional_1.map
AnnotationGAPDH, cyto, oxidative stress 24h, active, single subunit analysis, class4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: GAPDH, cyto, oxidative stress 24h, inactive, single subunit...

Fileemd_29663_additional_2.map
AnnotationGAPDH, cyto, oxidative stress 24h, inactive, single subunit analysis, class1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: GAPDH, cyto, oxidative stress 24h, nondefined, single subunit...

Fileemd_29663_additional_3.map
AnnotationGAPDH, cyto, oxidative stress 24h, nondefined, single subunit analysis, class2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: GAPDH, cyto, oxidative stress 24h, nondefined, single subunit...

Fileemd_29663_additional_4.map
AnnotationGAPDH, cyto, oxidative stress 24h, nondefined, single subunit analysis, class3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: GAPDH, cyto, oxidative stress 24h, nondefined, single subunit...

Fileemd_29663_additional_5.map
AnnotationGAPDH, cyto, oxidative stress 24h, nondefined, single subunit analysis, class5
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GAPDH, cyto, oxidative stress 24h, halfmap1, d2

Fileemd_29663_half_map_1.map
AnnotationGAPDH, cyto, oxidative stress 24h, halfmap1, d2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GAPDH, cyto, oxidative stress 24h, halfmap2, d2

Fileemd_29663_half_map_2.map
AnnotationGAPDH, cyto, oxidative stress 24h, halfmap2, d2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GAPDH

EntireName: GAPDHGlyceraldehyde 3-phosphate dehydrogenase
Components
  • Complex: GAPDHGlyceraldehyde 3-phosphate dehydrogenase
    • Protein or peptide: Glyceraldehyde-3-phosphate dehydrogenaseGlyceraldehyde 3-phosphate dehydrogenase

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Supramolecule #1: GAPDH

SupramoleculeName: GAPDH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Glyceraldehyde-3-phosphate dehydrogenase

MacromoleculeName: Glyceraldehyde-3-phosphate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
EC number: glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.967969 KDa
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statecell

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Sample preparation

BufferpH: 8
VitrificationCryogen name: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm 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 modelType of model: OTHER
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 598578

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