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- EMDB-0458: Structure of Dot1L-H2BK120ub nucleosome complex -

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Entry
Database: EMDB / ID: 0458
TitleStructure of Dot1L-H2BK120ub nucleosome complex
Map dataStructure of Dot1L-H2BK120ub nucleosome complex
SampleDot1L-H2BK120ub nucleosome complex (Class 2A)
  • Histone H3.2
  • Histone H4
  • Histone H2A type 1
  • Histone H2B 1.1
  • (nucleic-acidNucleic acid) x 2
  • Histone-lysine N-methyltransferase, H3 lysine-79 specificHistone methyltransferase
  • Ubiquitin
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-C / 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
SourceHomo sapiens (human) / Xenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsAnderson CJ / Baird MR / Hsu A / Barbour EH / Koyama Y / Borgnia MJ / McGinty RK
CitationJournal: Cell Rep / Year: 2019
Title: Structural Basis for Recognition of Ubiquitylated Nucleosome by Dot1L Methyltransferase.
Authors: Cathy J Anderson / Matthew R Baird / Allen Hsu / Emily H Barbour / Yuka Koyama / Mario J Borgnia / Robert K McGinty
Abstract: Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of ...Histone H3 lysine 79 (H3K79) methylation is enriched on actively transcribed genes, and its misregulation is a hallmark of leukemia. Methylation of H3K79, which resides on the structured disk face of the nucleosome, is mediated by the Dot1L methyltransferase. Dot1L activity is part of a trans-histone crosstalk pathway, requiring prior histone H2B ubiquitylation of lysine 120 (H2BK120ub) for optimal activity. However, the molecular details describing both how Dot1L binds to the nucleosome and why Dot1L is activated by H2BK120 ubiquitylation are unknown. Here, we present the cryoelectron microscopy (cryo-EM) structure of Dot1L bound to a nucleosome reconstituted with site-specifically ubiquitylated H2BK120. The structure reveals that Dot1L engages the nucleosome acidic patch using a variant arginine anchor and occupies a conformation poised for methylation. In this conformation, Dot1L and ubiquitin interact directly through complementary hydrophobic surfaces. This study establishes a path to better understand Dot1L function in normal and leukemia cells.
Validation ReportPDB-ID: 6nn6

SummaryFull reportAbout validation report
DateDeposition: Jan 14, 2019 / Header (metadata) release: Feb 6, 2019 / Map release: Feb 13, 2019 / Last update: Feb 27, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nn6
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0458.map.gz (map file in CCP4 format, 143749 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
330 pix
1.08 Å/pix.
= 356.4 Å
330 pix
1.08 Å/pix.
= 356.4 Å
330 pix
1.08 Å/pix.
= 356.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.11526908 - 0.19447334
Average (Standard dev.)0.00020446771 (0.0037517084)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions330330330
Origin0.00.00.0
Limit329.0329.0329.0
Spacing330330330
CellA=B=C: 356.40002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z330330330
origin x/y/z0.0000.0000.000
length x/y/z356.400356.400356.400
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS330330330
D min/max/mean-0.1150.1940.000

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Supplemental data

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Sample components

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Entire Dot1L-H2BK120ub nucleosome complex (Class 2A)

EntireName: Dot1L-H2BK120ub nucleosome complex (Class 2A) / Number of components: 9

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Component #1: protein, Dot1L-H2BK120ub nucleosome complex (Class 2A)

ProteinName: Dot1L-H2BK120ub nucleosome complex (Class 2A) / Recombinant expression: No
MassTheoretical: 270 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Histone H3.2

ProteinName: Histone H3.2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 15.30393 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: 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 #4: 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 #5: protein, Histone H2B 1.1

ProteinName: Histone H2B 1.1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 13.642846 kDa
SourceSpecies: Xenopus laevis (African clawed frog)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: nucleic-acid, DNA (145-MER)

Nucleic-acidName: DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 44.520383 kDa
SourceSpecies: synthetic construct (others)

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Component #7: nucleic-acid, DNA (145-MER)

Nucleic-acidName: DNA (145-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(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)
MassTheoretical: 44.99166 kDa
SourceSpecies: synthetic construct (others)

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Component #8: 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: 47.462039 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #9: protein, Ubiquitin

ProteinName: Ubiquitin / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 8.91118 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.93 mg/ml / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %

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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: 48 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1000

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 39558
3D reconstructionSoftware: RELION / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 1NW3, 1UBQ, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0, 3LZ0
Chain ID: A, A, A, B, C, D, E, F, G, H, I, J
Output model

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