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- EMDB-1878: CryoEM structure of the remodelling factor ISW1a bound to a monon... -

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Basic information

Entry
Database: EMDB / ID: EMD-1878
TitleCryoEM structure of the remodelling factor ISW1a bound to a mononucleosome (45N0)
Map dataSurface rendering of 2x45N0-ISW1a (delta ATPase)
Sample
  • Sample: Chromatin remodelling factor ISW1a (delta ATPase) bound to a mononucleosome (with a single 45 bp extension). This complex then forms a dimer.
  • Protein or peptide: Nucleosome
  • Protein or peptide: ISW1a
KeywordsChromatin remodelling factor / ISW1a / ISWI / nucleosome
Biological speciesXenopus laevis (African clawed frog) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.0 Å
AuthorsFrouws TD / Richmond TJ
CitationJournal: Nature / Year: 2011
Title: Structure and mechanism of the chromatin remodelling factor ISW1a.
Authors: Kazuhiro Yamada / Timothy D Frouws / Brigitte Angst / Daniel J Fitzgerald / Carl DeLuca / Kyoko Schimmele / David F Sargent / Timothy J Richmond /
Abstract: Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors ...Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.
History
DepositionFeb 16, 2011-
Header (metadata) releaseFeb 18, 2011-
Map releaseApr 21, 2011-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1878imag...

Downloads & links

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Map

FileDownload / File: emd_1878.map.gz / Format: CCP4 / Size: 17.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSurface rendering of 2x45N0-ISW1a (delta ATPase)
Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.0651294 - 0.167442
Average (Standard dev.)-0.0000352799 (±0.00979647)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 467.04 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z168168168
origin x/y/z-0.000-0.000-0.000
length x/y/z467.040467.040467.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS168168168
D min/max/mean-0.0650.167-0.000

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Supplemental data

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Sample components

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Entire : Chromatin remodelling factor ISW1a (delta ATPase) bound to a mono...

EntireName: Chromatin remodelling factor ISW1a (delta ATPase) bound to a mononucleosome (with a single 45 bp extension). This complex then forms a dimer.
Components
  • Sample: Chromatin remodelling factor ISW1a (delta ATPase) bound to a mononucleosome (with a single 45 bp extension). This complex then forms a dimer.
  • Protein or peptide: Nucleosome
  • Protein or peptide: ISW1a

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Supramolecule #1000: Chromatin remodelling factor ISW1a (delta ATPase) bound to a mono...

SupramoleculeName: Chromatin remodelling factor ISW1a (delta ATPase) bound to a mononucleosome (with a single 45 bp extension). This complex then forms a dimer.
type: sample / ID: 1000
Details: 45 bp segments bridge 2 DNA binding sites across the 2-fold axis, resulting in the auto-dimerisation.
Oligomeric state: Dimer / Number unique components: 2
Molecular weightTheoretical: 716 KDa

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Macromolecule #1: Nucleosome

MacromoleculeName: Nucleosome / type: protein_or_peptide / ID: 1 / Name.synonym: Nucleosome (45N0)
Details: Recombinant Xenopus histones H2A,H2B,H3,H4 are reconstituted onto a nucleosome containing the '601' positioning sequence and a single 45 bp extension.
Recombinant expression: Yes
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 250 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #2: ISW1a

MacromoleculeName: ISW1a / type: protein_or_peptide / ID: 2 / Name.synonym: Remodelling factor
Details: Recombinant Yeast ISW1a (delta ATPase) is expressed in the MultiBac system
Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 120 MDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

Concentration3.5 mg/mL
BufferpH: 7.5 / Details: 10mM TrisCl, 10mM KCl, 0,1 mM EDTA
GridDetails: C-flat 224
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK II / Details: Vitrification instrument: Vitribot MKII / Method: Offset 0, Blot 4 sec, Drain 1 sec

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 107520 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 100 K
Detailslow dose
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 14 µm / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Class averages
Final two d classificationNumber classes: 45
Final angle assignmentDetails: Theta 90, phi 180
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: Reconstructed with imposed 2-fold symmetry / Number images used: 4217
DetailsParticles manually picked with EMAN BOXER.

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Atomic model buiding 1

Initial modelPDB ID:

1kx5

SoftwareName: Chimera
DetailsManually fit using Chimera and "sym" command to impose 2-fold symmetry. Missing DNA segments were manually built in.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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