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- EMDB-0459: Structure of Dot1L-H2BK120ub nucleosome complex -

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Basic information

Entry
Database: EMDB / ID: 0459
TitleStructure of Dot1L-H2BK120ub nucleosome complex
Map data
SampleDot1L-H2BK120ub nucleosome complex (Classes 3 and 4):
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 3.5 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.
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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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0459.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-0.114047036 - 0.2319511
Average (Standard dev.)0.00019831856 (0.0039361087)
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-0.1140.2320.000

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Supplemental data

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Sample components

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Entire Dot1L-H2BK120ub nucleosome complex (Classes 3 and 4)

EntireName: Dot1L-H2BK120ub nucleosome complex (Classes 3 and 4) / Number of components: 1

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Component #1: protein, Dot1L-H2BK120ub nucleosome complex (Classes 3 and 4)

ProteinName: Dot1L-H2BK120ub nucleosome complex (Classes 3 and 4) / Recombinant expression: No
MassTheoretical: 270 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: 101430
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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