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- PDB-6nog: Poised-state Dot1L bound to the H2B-Ubiquitinated nucleosome -

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Basic information

Entry
Database: PDB / ID: 6nog
TitlePoised-state Dot1L bound to the H2B-Ubiquitinated nucleosome
Components
  • (601 DNA Strand ...) x 2
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone H3.2
  • Histone H4
  • Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
  • Ubiquitin
KeywordsSTRUCTURAL PROTEIN/TRANSFERASE/DNA / Ubiquitin / Nucleosome / Methyltransferase / STRUCTURAL PROTEIN-TRANSFERASE-DNA complex
Function / homology
Function and homology information


: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / histone methyltransferase activity / heterochromatin formation / telomere organization ...: / histone H3K79 trimethyltransferase activity / [histone H3]-lysine79 N-trimethyltransferase / histone H3K79 methyltransferase activity / regulation of transcription regulatory region DNA binding / regulation of receptor signaling pathway via JAK-STAT / histone H3 methyltransferase activity / histone methyltransferase activity / heterochromatin formation / telomere organization / DNA damage checkpoint signaling / PKMTs methylate histone lysines / structural constituent of chromatin / nucleosome / gene expression / RNA polymerase II-specific DNA-binding transcription factor binding / nucleic acid binding / transcription coactivator activity / protein heterodimerization activity / DNA repair / intracellular membrane-bounded organelle / positive regulation of cell population proliferation / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / nucleoplasm / nucleus / cytoplasm
Similarity search - Function
434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal Domain) / Histone H2B signature. ...434 Repressor (Amino-terminal Domain) - #60 / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / Histone H3-K79 methyltransferase / Histone methylation protein DOT1 / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Histone, subunit A / Histone, subunit A / 434 Repressor (Amino-terminal Domain) / 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 / Vaccinia Virus protein VP39 / 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. / Ubiquitin-like domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Ubiquitin B / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Histone-lysine N-methyltransferase, H3 lysine-79 specific
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsWorden, E.J. / Hoffmann, N.A. / Wolberger, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM095822 United States
CitationJournal: Cell / Year: 2019
Title: Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L.
Authors: Evan J Worden / Niklas A Hoffmann / Chad W Hicks / Cynthia Wolberger /
Abstract: Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is ...Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.
History
DepositionJan 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 27, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-0468
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Histone H3.2
E: Histone H3.2
B: Histone H4
F: Histone H4
C: Histone H2A type 1
G: Histone H2A type 1
D: Histone H2B 1.1
H: Histone H2B 1.1
L: Ubiquitin
K: Histone-lysine N-methyltransferase, H3 lysine-79 specific
I: 601 DNA Strand 1
J: 601 DNA Strand 2


Theoretical massNumber of molelcules
Total (without water)254,68812
Polymers254,68812
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56820 Å2
ΔGint-422 kcal/mol
Surface area99750 Å2

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Components

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Protein , 6 types, 10 molecules AEBFCGDHLK

#1: Protein Histone H3.2


Mass: 15303.930 Da / Num. of mol.: 2 / Mutation: G102A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P84233
#2: Protein Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P62799
#3: Protein Histone H2A type 1


Mass: 13978.241 Da / Num. of mol.: 2 / Mutation: G99R, A123S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P06897
#4: Protein Histone H2B 1.1 / H2B1.1


Mass: 13498.715 Da / Num. of mol.: 2 / Mutation: S32T, K120C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli (E. coli) / References: UniProt: P02281
#5: Protein Ubiquitin /


Mass: 9036.393 Da / Num. of mol.: 1 / Mutation: G76C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: J3QS39
#6: Protein Histone-lysine N-methyltransferase, H3 lysine-79 specific / Histone methyltransferase / DOT1-like protein / Histone H3-K79 methyltransferase / H3-K79-HMTase / Lysine N-methyltransferase 4 / Dot1L


Mass: 47432.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DOT1L, KIAA1814, KMT4 / Plasmid: pET32a / Production host: Escherichia coli (E. coli)
References: UniProt: Q8TEK3, histone-lysine N-methyltransferase

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601 DNA Strand ... , 2 types, 2 molecules IJ

#7: DNA chain 601 DNA Strand 1


Mass: 44825.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli)
#8: DNA chain 601 DNA Strand 2


Mass: 45305.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Poised state Dot1L in complex with the H2B-Ub nucleosomeCOMPLEX1:1 complex of Dot1L bound to the nucleosomeall0MULTIPLE SOURCES
2Dot1LCOMPLEX#61RECOMBINANT
3H2B-Ub nucleosomeCOMPLEX#1-#5, #7-#81MULTIPLE SOURCES
4histone coreCOMPLEX#1-#43RECOMBINANT
5ubiquitinCOMPLEX#53RECOMBINANT
6DNACOMPLEX#7-#83RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Xenopus laevis (African clawed frog)8355
35Homo sapiens (human)9606
46synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
12Escherichia coli (E. coli)562pET32a
24Escherichia coli (E. coli)562pET3a
35Escherichia coli (E. coli)562
46Escherichia coli (E. coli)562pST55-16x601
Buffer solutionpH: 7.5
Details: Solutions were prepared on the day of freezing and filtered though a 0.2 um filter prior to use.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaClSodium chloride1
22 mMdithiothreitolC4H10O2S21
320 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Crosslinked with glutaraldehyde
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot once for 3.5 seconds before freezing

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2267 / Details: 3 exposures per hole
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1cisTEM1.0.0particle selection
2Latitudeimage acquisition
4CTFFIND4.1CTF correction
9RELION2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 578660
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108658 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
11KX3

1kx3

A1KX31
51KX3

1kx3

B1KX31
61KX3

1kx3

C1KX31
71KX3

1kx3

D1KX31
81KX3

1kx3

E1KX31
91KX3

1kx3

F1KX31
101KX3

1kx3

G1KX31
111KX3

1kx3

H1KX31
23QOW

3qow

K3QOW2
31UBQ

1ubq

L1UBQ3
43MVD

3mvd

I3MVD4
123MVD

3mvd

J3MVD4

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