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- EMDB-0655: Structural basis of Dot1L stimulation by histone H2B lysine 120 u... -

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Basic information

Entry
Database: EMDB / ID: EMD-0655
TitleStructural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. 4.9A reconstruction of Dot1L on unmodified nucleosome
Map dataCryoSparc reconstruction, unsharpened. This density is rotated to fit with the final model.
Sample
  • Complex: Cryo-EM structure of human Dot1L bound to unmodified nucleosome at 4.9A resolution
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsValencia-Sanchez MI / De Ioannes P / Wang M / Vasilyev N / Chen R / Nudler E / Armache J-P / Armache K-J
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute5R01GM115882-03 United States
Other privateDavid and Lucille Packard Foundation United States
CitationJournal: Mol Cell / Year: 2019
Title: Structural Basis of Dot1L Stimulation by Histone H2B Lysine 120 Ubiquitination.
Authors: Marco Igor Valencia-Sánchez / Pablo De Ioannes / Miao Wang / Nikita Vasilyev / Ruoyu Chen / Evgeny Nudler / Jean-Paul Armache / Karim-Jean Armache /
Abstract: The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of ...The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.
History
DepositionMar 9, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 2019-
UpdateJun 19, 2019-
Current statusJun 19, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0655.png

Downloads & links

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Map

FileDownload / File: emd_0655.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoSparc reconstruction, unsharpened. This density is rotated to fit with the final model.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 256 pix.
= 311.194 Å		1.22 Å/pix.
x 256 pix.
= 311.194 Å		1.22 Å/pix.
x 256 pix.
= 311.194 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.21787588 - 0.837591
Average (Standard dev.)-0.0010382133 (±0.039305508)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21560156251.21560156251.2156015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z311.194311.194311.194
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.2180.838-0.001

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Supplemental data

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Additional map: CryoSparc reconstruction, mildly sharpened. This density is rotated...

Fileemd_0655_additional.map
AnnotationCryoSparc reconstruction, mildly sharpened. This density is rotated to fit with the final model.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoSparc half-map1. This map is in its original...

Fileemd_0655_half_map_1.map
AnnotationCryoSparc half-map1. This map is in its original position, not in line with the final model
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoSparc half-map2. This map is in its original...

Fileemd_0655_half_map_2.map
AnnotationCryoSparc half-map2. This map is in its original position, not in line with the final model
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of human Dot1L bound to unmodified nucleosome a...

EntireName: Cryo-EM structure of human Dot1L bound to unmodified nucleosome at 4.9A resolution
Components
  • Complex: Cryo-EM structure of human Dot1L bound to unmodified nucleosome at 4.9A resolution

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Supramolecule #1: Cryo-EM structure of human Dot1L bound to unmodified nucleosome a...

SupramoleculeName: Cryo-EM structure of human Dot1L bound to unmodified nucleosome at 4.9A resolution
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK I
Details: 3 ul of Dot1L-nucleosome complexes were applied to a glow discharged Quantifoil holey carbon grid (1.2 um hole size, 200 mesh), blotted in a Vitrobot Mark III (FEI Company) using 1.5 seconds ...Details: 3 ul of Dot1L-nucleosome complexes were applied to a glow discharged Quantifoil holey carbon grid (1.2 um hole size, 200 mesh), blotted in a Vitrobot Mark III (FEI Company) using 1.5 seconds blotting at 100% humidity, and then plunge-frozen in liquid ethane cooled by liquid nitrogen..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Sampling interval: 5.0 µm / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsFrames were motion corrected using MotionCor2 with dose-weighting
Particle selectionNumber selected: 705378
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL / In silico model: Generated using cryosparc ab initio run
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 107368
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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