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- PDB-6nj9: Active state Dot1L bound to the H2B-Ubiquitinated nucleosome, 2-t... -

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Basic information

Entry
Database: PDB / ID: 6nj9
TitleActive state Dot1L bound to the H2B-Ubiquitinated nucleosome, 2-to-1 complex
Components
  • 601 DNA Strand 1
  • 601 DNA strand 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 / homologyHistone H4, conserved site / Histone H2A/H2B/H3 / Histone H3/CENP-A / Histone H2B / Ubiquitin domain / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone-fold / Histone H2A, C-terminal domain ...Histone H4, conserved site / Histone H2A/H2B/H3 / Histone H3/CENP-A / Histone H2B / Ubiquitin domain / Histone H4 / Histone H2A / TATA box binding protein associated factor (TAF) / Histone-fold / Histone H2A, C-terminal domain / Histone H2A conserved site / Ubiquitin conserved site / Ubiquitin / Histone H3-K79 methyltransferase, metazoa / Histone-lysine N-methyltransferase DOT1 domain / S-adenosyl-L-methionine-dependent methyltransferase / Ubiquitin-like domain superfamily / CENP-T/Histone H4, histone fold / Core histone H2A/H2B/H3/H4 / Ubiquitin family / Ubiquitin domain signature. / PKMTs methylate histone lysines / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Ubiquitin domain profile. / Histone H3 signature 2. / Histone H2B signature. / Histone H3 signature 1. / Histone H4 signature. / Histone H2A signature. / C-terminus of histone H2A / Centromere kinetochore component CENP-T histone fold / Histone methylation protein DOT1 / Histone H3-K79 methyltransferase / histone methyltransferase activity (H3-K79 specific) / regulation of transcription regulatory region DNA binding / histone H3-K79 methylation / histone methyltransferase activity / regulation of receptor signaling pathway via JAK-STAT / chromatin silencing at telomere / histone-lysine N-methyltransferase / histone-lysine N-methyltransferase activity / telomere organization / DNA-templated transcription, initiation / DNA damage checkpoint / nucleosome / chromosome, telomeric region / nucleosome assembly / intracellular membrane-bounded organelle / transcription factor binding / positive regulation of cell population proliferation / protein heterodimerization activity / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / nucleoplasm / nucleus / Polyubiquitin-B / Histone H2B 1.1 / Histone H2A type 1 / Histone H4 / Histone H3.2 / Histone-lysine N-methyltransferase, H3 lysine-79 specific
Function and homology information
Specimen sourceXenopus laevis (African clawed frog)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsWorden, E.J. / Hoffmann, N.A. / Wolberger, C.
Funding supportUnited States , 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM095822United 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 2, 2019 / Release: Feb 20, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Feb 20, 2019Structure modelrepositoryInitial release
1.1Feb 27, 2019Structure modelData collection / Database referencescitation / citation_author_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
1.2Mar 20, 2019Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)254,98213
Polyers254,58412
Non-polymers3981
Water0
1


  • idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, gel filtration
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)61770
ΔGint (kcal/M)-403
Surface area (Å2)97190

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Components

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Protein/peptide , 6 types, 10 molecules FBGCHDKLEA

#1: Protein/peptide Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2 / Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Rosetta2 / References: UniProt: P62799
#2: Protein/peptide Histone H2A type 1


Mass: 13978.241 Da / Num. of mol.: 2 / Mutation: G99R, A123S / Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Rosetta2 / References: UniProt: P06897
#3: Protein/peptide Histone H2B 1.1 / H2B1.1


Mass: 13498.715 Da / Num. of mol.: 2 / Mutation: S32T, K120C / Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pET3a / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Rosetta2 / References: UniProt: P02281
#4: Protein/peptide 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: (gene. exp.) Homo sapiens (human) / Gene: DOT1L, KIAA1814, KMT4 / Plasmid: pET32a / Production host: Escherichia coli (E. coli)
References: UniProt: Q8TEK3, histone-lysine N-methyltransferase
#5: Protein/peptide Ubiquitin /


Mass: 9036.393 Da / Num. of mol.: 1 / Mutation: G76C / Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: J3QS39
#6: Protein/peptide Histone H3.2


Mass: 15251.830 Da / Num. of mol.: 2 / Mutation: G102A, K79Nle, M90Nle, M120Nle / Source: (gene. exp.) Xenopus laevis (African clawed frog) / Plasmid: pQE-81L / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): Rosetta2 / References: UniProt: P84233

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DNA chain , 2 types, 2 molecules IJ

#7: DNA chain 601 DNA Strand 1


Mass: 44825.559 Da / Num. of mol.: 1 / Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli) / Strain (production host): Xl1-blue
#8: DNA chain 601 DNA strand 2


Mass: 45305.852 Da / Num. of mol.: 1 / Source: (gene. exp.) synthetic construct (others) / Plasmid: pST55-16x601 / Production host: Escherichia coli (E. coli) / Strain (production host): Xl1-blue

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Non-polymers , 1 types, 1 molecules

#9: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE


Mass: 398.437 Da / Num. of mol.: 1 / Formula: C15H22N6O5S / S-Adenosyl methionine

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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

Type: COMPLEX

IDNameDetailsEntity IDParent-IDSource
1Active state Dot1L in complex with the H2B-Ub nucleosome2:1 complex of Dot1L bound to the nucleosome1, 2, 3, 4, 5, 6, 7, 80MULTIPLE SOURCES
2Dot1L41RECOMBINANT
3H2H2B-Ub nucleosome1, 2, 3, 5, 6, 7, 81MULTIPLE SOURCES
4histone core1, 2, 3, 63RECOMBINANT
5ubiquitin53RECOMBINANT
6DNA7, 83RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDNcbi tax-IDOrganism
129606Homo sapiens (human)
248355Xenopus laevis (African clawed frog)
359606Homo sapiens (human)
4632630synthetic construct (others)
Source (recombinant)
IDEntity assembly-IDNcbi tax-IDOrganismStrain
12511693Escherichia coli BL21 (bacteria)Rosetta2
24562Escherichia coli (E. coli)
35562Escherichia coli (E. coli)
46562Escherichia coli (E. coli)Xl1-blue
Buffer solutionDetails: Solutions were prepared on the day of freezing and filtered though a 0.2 um filter prior to use.
pH: 7.5
Buffer component

Buffer-ID: 1

IDConc. (mg/ml)NameFormula
150 mMsodium chlorideNaCl
22 mMdithiothreitolC4H10O2S2
320 mMHEPESC8H18N2O4S
SpecimenConc.: 0.75 mg/ml / Details: Crosslinked with glutaraldehyde / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / 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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 microns
Specimen holderModel: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.2 sec. / Electron dose: 50 e/Å2 / Details: 3 exposures per hole / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2284
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2Latitudeimage acquisition
4CTFFIND4.1CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 654532
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 237780 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
11KX3A
51KX3B
61KX3C
71KX3D
81KX3E
91KX3F
101KX3G
111KX3H
21NW3K
31UBQL
43MVDI
123MVDJ

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