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- EMDB-0460: Structure of Dot1L-H2BK120ub nucleosome complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0460
TitleStructure of Dot1L-H2BK120ub nucleosome complex
Map dataNone
Sample
  • Complex: Dot1L-H2BK120ub nucleosome complex (Class 2A masked)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
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.
History
DepositionJan 14, 2019-
Header (metadata) releaseJan 23, 2019-
Map releaseFeb 13, 2019-
UpdateFeb 27, 2019-
Current statusFeb 27, 2019Processing site: RCSB / Status: Released

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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

emd_0460.png

Downloads & links

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Map

FileDownload / File: emd_0460.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.17673753 - 0.33332458
Average (Standard dev.)0.00020607935 (±0.0033715756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 356.40002 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS330330330
D min/max/mean-0.1770.3330.000

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Supplemental data

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Sample components

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Entire : Dot1L-H2BK120ub nucleosome complex (Class 2A masked)

EntireName: Dot1L-H2BK120ub nucleosome complex (Class 2A masked)
Components
  • Complex: Dot1L-H2BK120ub nucleosome complex (Class 2A masked)

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Supramolecule #1: Dot1L-H2BK120ub nucleosome complex (Class 2A masked)

SupramoleculeName: Dot1L-H2BK120ub nucleosome complex (Class 2A masked) / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 270 KDa

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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

Concentration0.93 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1000 / Average exposure time: 60.0 sec. / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 408526
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6esf

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 39558 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 39558
FSC plot (resolution estimation)

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