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- EMDB-8246: Structural model of 53BP1 bound to a ubiquitylated and methylated... -

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Basic information

Entry
Database: EMDB / ID: EMD-8246
TitleStructural model of 53BP1 bound to a ubiquitylated and methylated nucleosome, at 4.5 A resolution
Map data53BP1 bound to a ubiquitylated and methylated nucleosome
Sample
  • Complex: NCP-ubme/GST-53BP1 complex
    • Complex: NCP-ubme
      • Complex: Widom-601 DNA
        • DNA: DNA (145-MER)
        • DNA: DNA (145-MER)
      • Complex: Ubiquitylated methylated histone octamer
        • Complex: Histone H4kc20Me2
          • Protein or peptide: Histone H4
        • Complex: Histone H3
          • Protein or peptide: Histone H3.2
        • Complex: Histone H2B.1
          • Protein or peptide: Histone H2B type 1-C/E/F/G/I
        • Complex: Histone H2A.1 K13RK36R
          • Protein or peptide: Histone H2A type 1
        • Complex: Ubiquitin
    • Complex: GST-53BP1
      • Protein or peptide: Tumor suppressor p53-binding protein 1
      • Protein or peptide: Ubiquitin
Function / homology
Function and homology information


ubiquitin-modified histone reader activity / positive regulation of isotype switching / cellular response to X-ray / double-strand break repair via classical nonhomologous end joining / DNA repair complex / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / positive regulation of protein monoubiquitination / mitochondrion transport along microtubule / fat pad development ...ubiquitin-modified histone reader activity / positive regulation of isotype switching / cellular response to X-ray / double-strand break repair via classical nonhomologous end joining / DNA repair complex / hypothalamus gonadotrophin-releasing hormone neuron development / female meiosis I / positive regulation of protein monoubiquitination / mitochondrion transport along microtubule / fat pad development / negative regulation of double-strand break repair via homologous recombination / telomeric DNA binding / female gonad development / seminiferous tubule development / male meiosis I / SUMOylation of transcription factors / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / regulation of proteasomal protein catabolic process / Replacement of protamines by nucleosomes in the male pronucleus / energy homeostasis / Packaging Of Telomere Ends / regulation of neuron apoptotic process / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Maturation of protein E / Maturation of protein E / Deposition of new CENPA-containing nucleosomes at the centromere / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / Endosomal Sorting Complex Required For Transport (ESCRT) / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Regulation of FZD by ubiquitination / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Negative regulation of FLT3 / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / methylated histone binding / APC/C:Cdc20 mediated degradation of Cyclin B / Inhibition of DNA recombination at telomere / Meiotic synapsis / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / APC-Cdc20 mediated degradation of Nek2A / PINK1-PRKN Mediated Mitophagy / TRAF6-mediated induction of TAK1 complex within TLR4 complex / InlA-mediated entry of Listeria monocytogenes into host cells / Pexophagy / Regulation of innate immune responses to cytosolic DNA / VLDLR internalisation and degradation / histone reader activity / Downregulation of ERBB2:ERBB3 signaling / NRIF signals cell death from the nucleus / Activated NOTCH1 Transmits Signal to the Nucleus / Translesion synthesis by REV1 / NF-kB is activated and signals survival / RNA Polymerase I Promoter Opening / Regulation of PTEN localization / Translesion synthesis by POLK / Regulation of BACH1 activity / Assembly of the ORC complex at the origin of replication / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / MAP3K8 (TPL2)-dependent MAPK1/3 activation / TICAM1, RIP1-mediated IKK complex recruitment / Downregulation of TGF-beta receptor signaling / Josephin domain DUBs / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / DNA methylation / Regulation of activated PAK-2p34 by proteasome mediated degradation / InlB-mediated entry of Listeria monocytogenes into host cell / IKK complex recruitment mediated by RIP1 / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / neuron projection morphogenesis / Condensation of Prophase Chromosomes / regulation of mitochondrial membrane potential / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / HCMV Late Events / Chromatin modifications during the maternal to zygotic transition (MZT) / replication fork / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / SIRT1 negatively regulates rRNA expression / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Autodegradation of Cdh1 by Cdh1:APC/C / innate immune response in mucosa / TNFR1-induced NF-kappa-B signaling pathway / PRC2 methylates histones and DNA
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
TP53-binding protein 1 / Histone H2A type 1 / Polyubiquitin-B / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.2 / TP53-binding protein 1
Similarity search - Component
Biological speciesunclassified (unknown) / synthetic construct (others) / Homo sapiens (human) / Xenopus laevis (African clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.54 Å
AuthorsBenlekbir S / Wilson MD / Sicheri F / Rubinstein JL / Durocher D
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FDN143343 Canada
Canadian Institutes of Health Research (CIHR)FDN143277 Canada
Canadian Institutes of Health Research (CIHR)MOP81294 Canada
CitationJournal: Nature / Year: 2016
Title: The structural basis of modified nucleosome recognition by 53BP1.
Abstract: DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases ...DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.
History
DepositionJul 11, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateJan 15, 2020-
Current statusJan 15, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kgf
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8246.png

Downloads & links

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Map

FileDownload / File: emd_8246.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation53BP1 bound to a ubiquitylated and methylated nucleosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.45 Å/pix.
x 128 pix.
= 185.6 Å		1.45 Å/pix.
x 128 pix.
= 185.6 Å		1.45 Å/pix.
x 128 pix.
= 185.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.45 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.08742182 - 0.17725106
Average (Standard dev.)0.0013958213 (±0.012893648)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 185.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z185.600185.600185.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0870.1770.001

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Supplemental data

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Sample components

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Entire : NCP-ubme/GST-53BP1 complex

EntireName: NCP-ubme/GST-53BP1 complex
Components
  • Complex: NCP-ubme/GST-53BP1 complex
    • Complex: NCP-ubme
      • Complex: Widom-601 DNA
        • DNA: DNA (145-MER)
        • DNA: DNA (145-MER)
      • Complex: Ubiquitylated methylated histone octamer
        • Complex: Histone H4kc20Me2
          • Protein or peptide: Histone H4
        • Complex: Histone H3
          • Protein or peptide: Histone H3.2
        • Complex: Histone H2B.1
          • Protein or peptide: Histone H2B type 1-C/E/F/G/I
        • Complex: Histone H2A.1 K13RK36R
          • Protein or peptide: Histone H2A type 1
        • Complex: Ubiquitin
    • Complex: GST-53BP1
      • Protein or peptide: Tumor suppressor p53-binding protein 1
      • Protein or peptide: Ubiquitin

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Supramolecule #1: NCP-ubme/GST-53BP1 complex

SupramoleculeName: NCP-ubme/GST-53BP1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Details: Single-particle electrocryomicroscopy structure of Tandem Tudor domain and UDR region of human 53BP1 bound to a recombinant ubiquitylated and methylated nucleosome core particle
Molecular weightExperimental: 318 KDa

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Supramolecule #2: NCP-ubme

SupramoleculeName: NCP-ubme / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#6, #9
Details: Modified nucleosome core particle, H2A enzymatically ubiquitylated on H2A K15, H4 chemically alkylated at K20C to create dimethyl lysine analog
Source (natural)Organism: unclassified (unknown)
Molecular weightTheoretical: 226 KDa

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Supramolecule #3: Widom-601 DNA

SupramoleculeName: Widom-601 DNA / type: complex / ID: 3 / Parent: 2 / Macromolecule list: #5-#6
Details: 145 bp fragment of Widom-601 strong nuclesome positioning sequence, gift from Curt Davey (Vasudevan et. al, 2010, J.Mol.Biol.)
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pUC57-8x145bp
Molecular weightTheoretical: 882.31 KDa

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Supramolecule #4: GST-53BP1

SupramoleculeName: GST-53BP1 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #7-#8
Details: 53BP1 Tandem Tudor domain and ubiquitin dependent recruitment region, artificially dimerized with Gluthaione-S-transferase (GST, not visible in structure)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2621
Molecular weightExperimental: 89.2 KDa

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Supramolecule #5: Ubiquitylated methylated histone octamer

SupramoleculeName: Ubiquitylated methylated histone octamer / type: complex / ID: 5 / Parent: 2 / Macromolecule list: #1-#4, #9
Source (natural)Organism: unclassified (unknown)
Molecular weightTheoretical: 138 KDa

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Supramolecule #6: Histone H4kc20Me2

SupramoleculeName: Histone H4kc20Me2 / type: complex / ID: 6 / Parent: 5 / Macromolecule list: #2 / Details: Dimethylated at position 20
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2349

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Supramolecule #7: Histone H3

SupramoleculeName: Histone H3 / type: complex / ID: 7 / Parent: 5 / Macromolecule list: #1
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD1874

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Supramolecule #8: Histone H2B.1

SupramoleculeName: Histone H2B.1 / type: complex / ID: 8 / Parent: 5 / Macromolecule list: #4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2644

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Supramolecule #9: Histone H2A.1 K13RK36R

SupramoleculeName: Histone H2A.1 K13RK36R / type: complex / ID: 9 / Parent: 5 / Macromolecule list: #3
Details: Crosslinked at H2AK15 to ubiquitin at ub G76 (isopeptide bond)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2647

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Supramolecule #10: Ubiquitin

SupramoleculeName: Ubiquitin / type: complex / ID: 10 / Parent: 5 / Macromolecule list: #9 / Details: Crosslinked to H2A K15 (isopeptide bond)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pDD2528

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 15.421101 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARTKQTARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSSA VMALQEASEA YLVGLFEDTN LCAIHAKRVT IMPKDIQLAR RIRGERA

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Details: cysteine alkylation at position 20 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 11.439511 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKGGKG LGKGGAKRHR (M2L)VLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEH AKR KTVTAMDVVY ALKRQGRTLY GFGG

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Macromolecule #3: Histone H2A type 1

MacromoleculeName: Histone H2A type 1 / type: protein_or_peptide / ID: 3
Details: Isopeptide amide crosslink between K15 of H2A and G76 of ubiquitin
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.163539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSGRGKQGGK ARARAKSRSS RAGLQFPVGR VHRLLRRGNY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIR NDEELNKLLG KVTIAQGGVL PNIQAVLLPK KTESHHKAKG K

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Macromolecule #4: Histone H2B type 1-C/E/F/G/I

MacromoleculeName: Histone H2B type 1-C/E/F/G/I / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.937213 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSV YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRST ITSREIQTAV RLLLPGELAK HAVSEGTKAV TKYTSSK

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Macromolecule #7: Tumor suppressor p53-binding protein 1

MacromoleculeName: Tumor suppressor p53-binding protein 1 / type: protein_or_peptide / ID: 7 / Details: full protein not modeled / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.376757 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
LTKAADISLD NLVEGKRKRR S

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Macromolecule #8: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 8.576831 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

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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

Concentration0.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
30.0 mMKClpotassium chloride
0.5 mMEDTAEthylenediaminetetraacetic acid
1.0 mMDTT

Details: High concentration NCP-ubme/GST-53BP1 complex at 200 mM salt was diluted just prior to grid freezing.
GridModel: electron micrsocopy sciences / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Details: Plunged into liquid ethane-propane (FEI VITROBOT MARK III).

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 25000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 2 / Number real images: 319 / Average exposure time: 0.5 sec. / Average electron dose: 36.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 174185
Details: Automatically picked from roughly 3000 particles using a manually picked template
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kx5


Details: Resized to the same pixel size as the data and low-pass filtered to 40 Angstrom resolution
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final reconstructionNumber classes used: 9 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 45361
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe atomic models of Widom-601 DNA (PDB ID 3LZ0), octameric histones (PDB ID 1KX5), ubiquitin (PDB ID 1UBI), and H4K20me2/53BP1 tandem Tudor domain (PDB ID 2IG0) were fitted without allowing flexibility into the 3D maps using UCSF Chimera. Segmentation was performed in UCSF Chimera. For the NCP-ubme structure the ubiquitin segmentation was further modified to remove obvious NCP density from the ubiquitin segment. The H2A/H2B sequence was mutated to the human H2AK13R/K36R and H2B manually in UCSF Chimera. A polyalanine model of the UDR was built within the UDR density in Coot, which compared well to predicted structures generated by Rosetta. The UDR model was mutated and fitted using UCSF Chimera, followed by iterative rounds of real-space refinement in PHENIX and model optimization in Coot. All figures were prepared in UCSF Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 207.5
Output model

PDB-5kgf:
Structural model of 53BP1 bound to a ubiquitylated and methylated nucleosome, at 4.5 A resolution

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Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

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