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- EMDB-29659: CryoEM structure of nuclear GAPDH under 8h Oxidative Stress -

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Basic information

Entry
Database: EMDB / ID: EMD-29659
TitleCryoEM structure of nuclear GAPDH under 8h Oxidative Stress
Map dataGAPDH,Nuclear Oxidation 8h,D2
Sample
  • Complex: GAPDH
    • Protein or peptide: Glyceraldehyde-3-phosphate dehydrogenase
KeywordsGAPDH / energy / cytosolic protein / TRANSFERASE
Function / homology
Function and homology information


peptidyl-cysteine S-trans-nitrosylation / Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / negative regulation of endopeptidase activity / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / killing by host of symbiont cells / aspartic-type endopeptidase inhibitor activity / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / Gluconeogenesis / Glycolysis / GAIT complex ...peptidyl-cysteine S-trans-nitrosylation / Transferases; Transferring nitrogenous groups; Transferring other nitrogenous groups / negative regulation of endopeptidase activity / glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) / killing by host of symbiont cells / aspartic-type endopeptidase inhibitor activity / glyceraldehyde-3-phosphate dehydrogenase (NAD+) (phosphorylating) activity / Gluconeogenesis / Glycolysis / GAIT complex / peptidyl-cysteine S-nitrosylase activity / 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 / antimicrobial humoral immune response mediated by antimicrobial peptide / disordered domain specific binding / NADP binding / microtubule cytoskeleton / neuron apoptotic process / nuclear membrane / microtubule binding / vesicle / killing of cells of another organism / positive regulation of canonical NF-kappaB signal transduction / negative regulation of translation / protein stabilization / ribonucleoprotein complex / intracellular membrane-bounded organelle / perinuclear region of cytoplasm / extracellular exosome / identical protein binding / nucleus / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Glyceraldehyde 3-phosphate dehydrogenase, active site / Glyceraldehyde 3-phosphate dehydrogenase active site. / Glyceraldehyde-3-phosphate dehydrogenase, type I / 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 (humans) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.17 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-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29659.png

Downloads & links

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Map

FileDownload / File: emd_29659.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGAPDH,Nuclear Oxidation 8h,D2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 240 pix.
= 200.4 Å		0.84 Å/pix.
x 240 pix.
= 200.4 Å		0.84 Å/pix.
x 240 pix.
= 200.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0091
Minimum - Maximum-0.008813469 - 0.038337857
Average (Standard dev.)0.000070306756 (±0.0019736248)
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,Nuclear Oxidation 8h,single subunit analysis,inactive

Fileemd_29659_additional_1.map
AnnotationGAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive
Projections & Slices
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Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive

Fileemd_29659_additional_2.map
AnnotationGAPDH,Nuclear Oxidation 8h,single subunit analysis,inactive
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Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined

Fileemd_29659_additional_3.map
AnnotationGAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
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Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined

Fileemd_29659_additional_4.map
AnnotationGAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
Projections & Slices
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Additional map: GAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined

Fileemd_29659_additional_5.map
AnnotationGAPDH,Nuclear Oxidation 8h,single subunit analysis,non defined
Projections & Slices
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Half map: GAPDH,Nuclear Oxidation 8h,D2,halfmap1

Fileemd_29659_half_map_1.map
AnnotationGAPDH,Nuclear Oxidation 8h,D2,halfmap1
Projections & Slices
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Half map: GAPDH,Nuclear Oxidation 8h,D2,halfmap2

Fileemd_29659_half_map_2.map
AnnotationGAPDH,Nuclear Oxidation 8h,D2,halfmap2
Projections & Slices
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Sample components

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Entire : GAPDH

EntireName: GAPDH
Components
  • Complex: GAPDH
    • Protein or peptide: Glyceraldehyde-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 (humans)

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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: 35.983969 KDa
Recombinant expressionOrganism: Homo (humans)
SequenceString: 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: 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 45.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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 modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 764164
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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