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- EMDB-0480: Active state Dot1L bound to the H2B-Ubiquitinated nucleosome, 1-t... -

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Entry
Database: EMDB / Id: 480
TitleActive state Dot1L bound to the H2B-Ubiquitinated nucleosome, 1-to-1 complex
Map dataSharpened map of the 1-to-1 complex between Dot1L and the ubiquitinated nucleosome
SampleActive state Dot1L in complex with the H2B-Ub nucleosome
  • Dot1L
  • H2B-Ub nucleosome
  • histone core
  • ubiquitin
  • DNA
  • (nucleic-acidNucleic acid) x 2
  • Histone H4
  • Histone H2A type 1
  • Histone H2B 1.1
  • Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
  • Ubiquitin
  • Histone H3.2
  • ligand
Func homologySCF(Skp2)-mediated degradation of p27/p21 / Vpu mediated degradation of CD4 / APC/C:Cdc20 mediated degradation of Cyclin B / Autodegradation of Cdh1 by Cdh1:APC/C / SCF-beta-TrCP mediated degradation of Emi1 / APC/C:Cdc20 mediated degradation of Securin / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Membrane binding and targetting of GAG proteins / Assembly Of The HIV Virion ...SCF(Skp2)-mediated degradation of p27/p21 / Vpu mediated degradation of CD4 / APC/C:Cdc20 mediated degradation of Cyclin B / Autodegradation of Cdh1 by Cdh1:APC/C / SCF-beta-TrCP mediated degradation of Emi1 / APC/C:Cdc20 mediated degradation of Securin / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Membrane binding and targetting of GAG proteins / Assembly Of The HIV Virion / APC-Cdc20 mediated degradation of Nek2A / Vif-mediated degradation of APOBEC3G / TICAM1, RIP1-mediated IKK complex recruitment / EGFR downregulation / TNFR2 non-canonical NF-kB pathway / Degradation of beta-catenin by the destruction complex / TCF dependent signaling in response to WNT / Downstream TCR signaling / NRIF signals cell death from the nucleus / p75NTR recruits signalling complexes / NF-kB is activated and signals survival / Regulation of activated PAK-2p34 by proteasome mediated degradation / NOTCH1 Intracellular Domain Regulates Transcription / DDX58/IFIH1-mediated induction of interferon-alpha/beta / NOD1/2 Signaling Pathway / Downregulation of TGF-beta receptor signaling / Histone-lysine N-methyltransferase DOT1 (EC 2.1.1.43) domain profile. / Centromere kinetochore component CENP-T histone fold / C-terminus of histone H2A / Histone H2A signature. / Histone H4 signature. / Ubiquitin domain signature. / Histone H3 signature 1. / Histone H2B signature. / Histone H3 signature 2. / Ubiquitin domain profile. / Translesion synthesis by REV1 / Budding and maturation of HIV virion / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / Activation of NF-kappaB in B cells / ISG15 antiviral mechanism / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / ER-Phagosome pathway / Downregulation of ERBB4 signaling / Spry regulation of FGF signaling / Downregulation of ERBB2:ERBB3 signaling / Activated NOTCH1 Transmits Signal to the Nucleus / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / Ubiquitin family / Dectin-1 mediated noncanonical NF-kB signaling / Degradation of AXIN / Degradation of DVL / Regulation of FZD by ubiquitination / Pink/Parkin Mediated Mitophagy / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Regulation of TNFR1 signaling / TNFR1-induced NFkappaB signaling pathway / Hedgehog ligand biogenesis / Hh mutants that don't undergo autocatalytic processing are degraded by ERAD / CLEC7A (Dectin-1) signaling / AUF1 (hnRNP D0) binds and destabilizes mRNA / Degradation of GLI1 by the proteasome / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / Hedgehog 'on' state / Negative regulation of FGFR1 signaling / Negative regulation of FGFR2 signaling / Negative regulation of FGFR3 signaling / Negative regulation of FGFR4 signaling / Translesion synthesis by POLK / Translesion synthesis by POLI / Asymmetric localization of PCP proteins / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Downregulation of SMAD2/3:SMAD4 transcriptional activity / FCERI mediated NF-kB activation / SMAD2/SMAD3:SMAD4 heterotrimer regulates transcription / Separation of Sister Chromatids / Oxidative Stress Induced Senescence / Senescence-Associated Secretory Phenotype (SASP) / Oncogene Induced Senescence / Regulation of PLK1 Activity at G2/M Transition / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Stimuli-sensing channels / Constitutive Signaling by NOTCH1 HD Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / activated TAK1 mediates p38 MAPK activation / NOTCH2 Activation and Transmission of Signal to the Nucleus / Regulation of innate immune responses to cytosolic DNA / PKMTs methylate histone lysines / Glycogen synthesis / Autodegradation of the E3 ubiquitin ligase COP1 / Deactivation of the beta-catenin transactivating complex / Myoclonic epilepsy of Lafora / ABC-family proteins mediated transport / Circadian Clock / TAK1 activates NFkB by phosphorylation and activation of IKKs complex / Histone methylation protein DOT1 / Core histone H2A/H2B/H3/H4
Function and homology information
SourceXenopus laevis (African clawed frog) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsWorden EJ / Hoffmann NA / Wolberger C
CitationJournal: Cell / Year: 2019
Title: Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L.
P-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 ReportPDB-ID: 6nqa

SummaryFull reportAbout validation report
DateDeposition: Jan 19, 2019 / Header (metadata) release: Feb 6, 2019 / Map release: Feb 20, 2019 / Last update: Mar 20, 2019

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Strvis

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nqa
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downlink

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Map

Fileemd_0480.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.06 Å/pix.
= 272.384 Å
256 pix
1.06 Å/pix.
= 272.384 Å
256 pix
1.06 Å/pix.
= 272.384 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density
Contour Level:0.0166 (by author), 0.0166 (movie #1):
Minimum - Maximum-0.07713457 - 0.12983139
Average (Standard dev.)0.00021672556 (0.0030338003)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.00.00.0
Limit255.0255.0255.0
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z272.384272.384272.384
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0770.1300.000

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Supplemental data

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Mask #1

Fileemd_0480_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Active state Dot1L in complex with the H2B-Ub nucleosome

EntireName: Active state Dot1L in complex with the H2B-Ub nucleosome
Details: 1:1 complex of Dot1L bound to the nucleosome / Number of components: 15

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Component #1: protein, Active state Dot1L in complex with the H2B-Ub nucleosome

ProteinName: Active state Dot1L in complex with the H2B-Ub nucleosome
Details: 1:1 complex of Dot1L bound to the nucleosome / Recombinant expression: No

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Component #2: protein, Dot1L

ProteinName: Dot1L / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET32a

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Component #3: protein, H2B-Ub nucleosome

ProteinName: H2B-Ub nucleosome / Recombinant expression: No

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Component #4: protein, histone core

ProteinName: histone core / Recombinant expression: No
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET3a,pQE-81L

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Component #5: protein, ubiquitin

ProteinName: ubiquitin / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pST55-16x601

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Component #7: nucleic-acid, 601 DNA Strand 1

Nucleic-acidName: 601 DNA Strand 1 / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DC)(DG)(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)(DC)(DG)(DA)(DT)
MassTheoretical: 44.825559 kDa
SourceSpecies: synthetic construct (others)

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Component #8: nucleic-acid, 601 DNA Strand 2

Nucleic-acidName: 601 DNA Strand 2 / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DC)(DG)(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)(DC)(DG)(DA)
MassTheoretical: 45.305852 kDa
SourceSpecies: synthetic construct (others)

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Component #9: protein, Histone H4

ProteinName: Histone H4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.263231 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #10: protein, Histone H2A type 1

ProteinName: Histone H2A type 1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.978241 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #11: protein, Histone H2B 1.1

ProteinName: Histone H2B 1.1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.498715 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #12: protein, Histone-lysine N-methyltransferase, H3 lysine-79 specific

ProteinName: Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 47.432012 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #13: protein, Ubiquitin

ProteinName: Ubiquitin / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.036393 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #14: protein, Histone H3.2

ProteinName: Histone H3.2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 15.25183 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #15: ligand, S-ADENOSYLMETHIONINE

LigandName: S-ADENOSYLMETHIONINES-Adenosyl methionine / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.398437 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.75 mg/ml
Buffer solution: Solutions were prepared on the day of freezing and filtered though a 0.2 um filter prior to use.
Ph: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 % / 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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2284 / Details: 3 exposures per hole

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 220681
3D reconstructionSoftware: RELION / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 1KX3, 1KX3, 1KX3, 1KX3, 1KX3, 1KX3, 1KX3, 1KX3, 1NW3, 1UBQ, 3MVD, 3MVD
Chain ID: A, B, C, D, E, F, G, H, K, L, I, J
Output model

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