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- EMDB-6738: Human HP1alpha-dinucleosome H3K9Me3 class A -

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Basic information

Entry
Database: EMDB / ID: EMD-6738
TitleHuman HP1alpha-dinucleosome H3K9Me3 class A
Map data
Sample
  • Complex: Human HP1alpha-dinucleosome H3K9Me3 complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.5 Å
AuthorsMachida S / Takizawa Y / Ishimaru M / Sekine S / Sugita Y / Nakayama J / Wolf M / Kurumizaka H
Funding support Japan, 3 items
OrganizationGrant numberCountry
JSPSJP25116002 Japan
JSPS16K18473 Japan
JSPSJP25250023 Japan
CitationJournal: Mol Cell / Year: 2018
Title: Structural Basis of Heterochromatin Formation by Human HP1.
Authors: Shinichi Machida / Yoshimasa Takizawa / Masakazu Ishimaru / Yukihiko Sugita / Satoshi Sekine / Jun-Ichi Nakayama / Matthias Wolf / Hitoshi Kurumizaka /
Abstract: Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. ...Heterochromatin plays important roles in transcriptional silencing and genome maintenance by the formation of condensed chromatin structures, which determine the epigenetic status of eukaryotic cells. The trimethylation of histone H3 lysine 9 (H3K9me3), a target of heterochromatin protein 1 (HP1), is a hallmark of heterochromatin formation. However, the mechanism by which HP1 folds chromatin-containing H3K9me3 into a higher-order structure has not been elucidated. Here we report the three-dimensional structure of the H3K9me3-containing dinucleosomes complexed with human HP1α, HP1β, and HP1γ, determined by cryogenic electron microscopy with a Volta phase plate. In the structures, two H3K9me3 nucleosomes are bridged by a symmetric HP1 dimer. Surprisingly, the linker DNA between the nucleosomes does not directly interact with HP1, thus allowing nucleosome remodeling by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The structure depicts the fundamental architecture of heterochromatin.
History
DepositionMay 22, 2017-
Header (metadata) releaseJan 31, 2018-
Map releaseJan 31, 2018-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 4.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6738.png

Downloads & links

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Map

FileDownload / File: emd_6738.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 256 pix.
= 355.84 Å		1.39 Å/pix.
x 256 pix.
= 355.84 Å		1.39 Å/pix.
x 256 pix.
= 355.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.39 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2 / Movie #1: 4.2
Minimum - Maximum-2.5719435 - 15.663911
Average (Standard dev.)0.00059624464 (±0.9999543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 355.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z355.840355.840355.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.57215.6640.001

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Supplemental data

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Sample components

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Entire : Human HP1alpha-dinucleosome H3K9Me3 complex

EntireName: Human HP1alpha-dinucleosome H3K9Me3 complex
Components
  • Complex: Human HP1alpha-dinucleosome H3K9Me3 complex

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Supramolecule #1: Human HP1alpha-dinucleosome H3K9Me3 complex

SupramoleculeName: Human HP1alpha-dinucleosome H3K9Me3 complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Recombinant plasmid: pH2A, pH2B, pH3.2K9C/C110A, pH4, pHP1a-pre
Molecular weightTheoretical: 500 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

Concentration.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris-HCl
1.0 mMDithiothreitolDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 100.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 9.33 kPa / Details: Gatan Solarus
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: 3 second blot, 2.5uL.
Detailssample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 100.0 K
Alignment procedureComa free - Residual tilt: 0.1 mrad
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: Gatan Quantum 968 / Energy filter - Lower energy threshold: -10 eV / Energy filter - Upper energy threshold: 10 eV
Detailsnanoprobe, parallel beam illumination
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 2329 / Average exposure time: 5.0 sec. / Average electron dose: 38.0 e/Å2
Details: Images were collected with a volta phase plate (VPP) in-focus
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 35971 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.62 mm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailsframe alignment and integration with motioncor2 incl. dose weighting
Particle selectionNumber selected: 409948
CTF correctionDetails: in-focus VPP data
Startup modelType of model: OTHER / Details: cylinder generated in SPIDER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 21793
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 6 / Avg.num./class: 22000 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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