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- EMDB-34028: cryo-EM structure of gammaH2AXK15ub-H4K20me2 nucleosome bound to 53BP1 -

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Basic information

Entry
Database: EMDB / ID: EMD-34028
Titlecryo-EM structure of gammaH2AXK15ub-H4K20me2 nucleosome bound to 53BP1
Map data
Sample
  • Complex: Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome
    • Protein or peptide: Histone H3.1Histone H3
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2AX
    • Protein or peptide: Histone H2B
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
    • Protein or peptide: Polyubiquitin-B
    • Protein or peptide: UDR motif of 53BP1
    • Protein or peptide: TP53-binding protein 1
Function / homology
Function and homology information


Chromatin modifying enzymes / epigenetic regulation of gene expression / telomere organization / RNA Polymerase I Promoter Opening / Interleukin-7 signaling / Assembly of the ORC complex at the origin of replication / DNA methylation / Condensation of Prophase Chromosomes / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) ...Chromatin modifying enzymes / epigenetic regulation of gene expression / telomere organization / RNA Polymerase I Promoter Opening / Interleukin-7 signaling / Assembly of the ORC complex at the origin of replication / DNA methylation / Condensation of Prophase Chromosomes / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / PRC2 methylates histones and DNA / Defective pyroptosis / HDACs deacetylate histones / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / NoRC negatively regulates rRNA expression / B-WICH complex positively regulates rRNA expression / HDMs demethylate histones / PKMTs methylate histone lysines / RMTs methylate histone arginines / Meiotic recombination / Pre-NOTCH Transcription and Translation / nucleosome assembly / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / nucleosome / gene expression / RUNX1 regulates transcription of genes involved in differentiation of HSCs / chromatin organization / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / Estrogen-dependent gene expression / nuclear body / cadherin binding / Amyloid fiber formation / protein heterodimerization activity / DNA damage response / protein-containing complex / DNA binding / extracellular exosome / extracellular region / nucleoplasm / membrane / nucleus
Similarity search - Function
: / BRCA1 C Terminus (BRCT) domain / Tumour suppressor p53-binding protein-1 Tudor domain / Tumour suppressor p53-binding protein-1 Tudor / : / : / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily ...: / BRCA1 C Terminus (BRCT) domain / Tumour suppressor p53-binding protein-1 Tudor domain / Tumour suppressor p53-binding protein-1 Tudor / : / : / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H2A / Histone 2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Ubiquitin conserved site / Ubiquitin domain / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Ubiquitin domain signature. / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold / Ubiquitin family / Ubiquitin homologues / Ubiquitin-like domain / Ubiquitin domain profile. / Ribosomal protein L2, domain 2 / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Histone H4 / Histone H2A / Tumor protein p53 binding protein 1 / Ubiquitin B / Histone H3.1 / Histone H2B type 1-K
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsAi HS / GuoChao C / Qingyue G / Ze-Bin T / Zhiheng D / Xin L / Fan Y / Ziyu X / Jia-Bin L / Changlin T / Liu L
Funding support China, 5 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)22137005, 22137005, 81621002, and 21621003 for L. L., 21977090 for J.B. L., and 21825703 for C. T. China
The Strategic Priority Research Program of Chinese Academy of SciencesXDB37000000 for C.T. China
The China Postdoctoral Science Foundation2021M691747 for G. C. C. China
The China National Postdoctoral Program for Innovative TalentsBH2340000159 for F.Y. China
Special funding from China Postdoctoral Science Foundation2022TQ0170 for H.S.A. China
CitationJournal: J Am Chem Soc / Year: 2022
Title: Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes.
Authors: Huasong Ai / Guo-Chao Chu / Qingyue Gong / Ze-Bin Tong / Zhiheng Deng / Xin Liu / Fan Yang / Ziyu Xu / Jia-Bin Li / Changlin Tian / Lei Liu /
Abstract: The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe ...The chemical synthesis of homogeneously modified histones is a powerful approach to quantitatively decipher how post-translational modifications (PTMs) modulate epigenetic events. Herein, we describe the expedient syntheses of a selection of phosphorylated and ubiquitinated H2AX proteins in a strategy integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation. These modified H2AX proteins were then used to discover that although H2AXS139 phosphorylation can enhance the binding of the DNA damage repair factor 53BP1 to either an unmodified nucleosome or that bearing a single H2AXK15ub or H4K20me2 modification, it augments 53BP1's binding only weakly to nucleosomes bearing both H2AXK15ub and H4K20me2. To better understand why such a trivalent additive effect is lacking, we solved the cryo-EM structure (3.38 Å) of the complex of 53BP1 with the H2AXK15ub/S139ph_H4K20me2 nucleosome, which showed that H2AXS139 phosphorylation distorts the interaction interface between ubiquitin and 53BP1's UDR motif. Our study revealed that there is redundancy in the interplay of multiple histone PTMs, which may be useful for controlling the dynamic distribution of effector proteins onto nucleosomes bearing different histone variants and PTMs in a time-dependent fashion, through specific cellular biochemical events.
History
DepositionAug 7, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateFeb 15, 2023-
Current statusFeb 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34028.png

Downloads & links

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Map

FileDownload / File: emd_34028.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.026431082 - 0.06723061
Average (Standard dev.)0.00025095665 (±0.0023460023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_34028_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34028_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34028_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome

EntireName: Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome
Components
  • Complex: Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome
    • Protein or peptide: Histone H3.1Histone H3
    • Protein or peptide: Histone H4
    • Protein or peptide: Histone H2AX
    • Protein or peptide: Histone H2B
    • DNA: DNA (145-MER)
    • DNA: DNA (145-MER)
    • Protein or peptide: Polyubiquitin-B
    • Protein or peptide: UDR motif of 53BP1
    • Protein or peptide: TP53-binding protein 1

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Supramolecule #1: Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome

SupramoleculeName: Complex of 53BP1(1484-1972) and gammaH2AXK15ub-H4K20me2 nucleosome
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Histone H3.1

MacromoleculeName: Histone H3.1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.762835 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KKPHRYRPGT VALREIRRYQ KSTELLIRKL PFQRLVREIA QDFKTDLRFQ SSAVMALQEA CEAYLVGLFE DTNLCAIHAK RVTIMPKDI QLARRIRGER A

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.409056 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #3: Histone H2AX

MacromoleculeName: Histone H2AX / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.194211 KDa
SequenceString:
ARAKACTRSS RAGLQFPVGR VHRLLRKGHY SERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIRN DEELNKLLG GVTIAQGGVL PNIQAVLLPK KTE

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Macromolecule #4: Histone H2B

MacromoleculeName: Histone H2B / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.764366 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
RKRSRKESYS VYVYKVLKQV HPDTGISSKA MGIMNSFVND IFERIAGEAS RLAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKA VTKYTSA

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Macromolecule #7: Polyubiquitin-B

MacromoleculeName: Polyubiquitin-B / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.519778 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRG

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Macromolecule #8: UDR motif of 53BP1

MacromoleculeName: UDR motif of 53BP1 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.075418 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KAADISLDNL VEGKRKRR

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Macromolecule #9: TP53-binding protein 1

MacromoleculeName: TP53-binding protein 1 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.447231 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SFVGLRVVAK WSSNGYFYSG KITRDVGAGK YKLLFDDGYE CDVLGKDILL CDPIPLDTEV TALSEDEYFS AGVVKGHRKE SGELYYSIE KEGQRKWYKR MAVILSLEQG NRLREQYGL

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Macromolecule #5: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.520383 KDa
SequenceString: (DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC) ...String:
(DA)(DT)(DC)(DA)(DG)(DA)(DA)(DT)(DC)(DC) (DC)(DG)(DG)(DT)(DG)(DC)(DC)(DG)(DA)(DG) (DG)(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DA) (DT)(DT)(DG)(DG)(DT)(DC)(DG)(DT)(DA)(DG) (DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA) (DG)(DC)(DA)(DC)(DC)(DG)(DC)(DT)(DT)(DA) (DA)(DA) (DC)(DG)(DC)(DA)(DC)(DG)(DT) (DA)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT)(DC) (DC)(DC)(DC) (DC)(DG)(DC)(DG)(DT)(DT) (DT)(DT)(DA)(DA)(DC)(DC)(DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG) (DG)(DA)(DT)(DT)(DA) (DC)(DT)(DC)(DC)(DC)(DT)(DA)(DG)(DT)(DC) (DT)(DC)(DC)(DA)(DG) (DG)(DC)(DA)(DC) (DG)(DT)(DG)(DT)(DC)(DA)(DG)(DA)(DT)(DA) (DT)(DA)(DT)(DA)(DC)(DA) (DT)(DC)(DG) (DA)(DT)

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Macromolecule #6: DNA (145-MER)

MacromoleculeName: DNA (145-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 44.99166 KDa
SequenceString: (DA)(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DA) (DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG) (DT)(DA)(DA)(DT)(DC)(DC) ...String:
(DA)(DT)(DC)(DG)(DA)(DT)(DG)(DT)(DA)(DT) (DA)(DT)(DA)(DT)(DC)(DT)(DG)(DA)(DC)(DA) (DC)(DG)(DT)(DG)(DC)(DC)(DT)(DG)(DG) (DA)(DG)(DA)(DC)(DT)(DA)(DG)(DG)(DG)(DA) (DG) (DT)(DA)(DA)(DT)(DC)(DC)(DC)(DC) (DT)(DT)(DG)(DG)(DC)(DG)(DG)(DT)(DT)(DA) (DA)(DA) (DA)(DC)(DG)(DC)(DG)(DG)(DG) (DG)(DG)(DA)(DC)(DA)(DG)(DC)(DG)(DC)(DG) (DT)(DA)(DC) (DG)(DT)(DG)(DC)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DC)(DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG) (DA)(DG)(DC)(DT)(DG) (DT)(DC)(DT)(DA)(DC)(DG)(DA)(DC)(DC)(DA) (DA)(DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG) (DC)(DC)(DT)(DC)(DG)(DG)(DC)(DA)(DC)(DC) (DG)(DG)(DG)(DA)(DT)(DT) (DC)(DT)(DG) (DA)(DT)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 90830

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