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- EMDB-2224: Negative stain microscopy of a dimer of Actin-related protein 8 (... -

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Basic information

Entry
Database: EMDB / ID: EMD-2224
TitleNegative stain microscopy of a dimer of Actin-related protein 8 (Arp8) from S. cerevisiae.
Map dataNegative stain reconstruction of a dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
Sample
  • Sample: Dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
  • Protein or peptide: Actin-like protein ARP8
KeywordsChromatin remodelling / INO80 complex / Actin related protein / Arp8
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsBose DA / Saravanan M / Wuerges J / McCormack EA / Cook NJ / Zhang X / Wigley DB
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex.
Authors: Matheshwaran Saravanan / Jochen Wuerges / Daniel Bose / Elizabeth A McCormack / Nicola J Cook / Xiaodong Zhang / Dale B Wigley /
Abstract: Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal ...Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.
History
DepositionOct 21, 2012-
Header (metadata) releaseNov 7, 2012-
Map releaseDec 12, 2012-
UpdateDec 26, 2012-
Current statusDec 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2224.tif

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Map

FileDownload / File: emd_2224.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of a dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
Voxel sizeX=Y=Z: 3.52 Å
Density
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.05438939 - 0.10891778
Average (Standard dev.)0.00004303 (±0.00205883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-63-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.523.523.52
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z450.560450.560450.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-64-63-64
NC/NR/NS128128128
D min/max/mean-0.0540.1090.000

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Supplemental data

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Sample components

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Entire : Dimeric form of full-length Actin Related Protein 8 (Arp8) from S...

EntireName: Dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
Components
  • Sample: Dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
  • Protein or peptide: Actin-like protein ARP8

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Supramolecule #1000: Dimeric form of full-length Actin Related Protein 8 (Arp8) from S...

SupramoleculeName: Dimeric form of full-length Actin Related Protein 8 (Arp8) from Saccharomyces cerevisiae.
type: sample / ID: 1000 / Oligomeric state: dimer / Number unique components: 1
Molecular weightExperimental: 217 KDa / Theoretical: 200 KDa
Method: Multi angle light scattering (MALS) (0.231MDa) and Analytical ultracentrifugation (AUC) (0.217MDa)

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Macromolecule #1: Actin-like protein ARP8

MacromoleculeName: Actin-like protein ARP8 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Nucleus
Molecular weightTheoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
StainingType: NEGATIVE
Details: 2 ul of ~0.05mg/ml Full length Arp8 was applied to glow-discharged continuous carbon grids (TAAB). Sample was adsorbed for 20s, then stained with 2% w/v uranyl acetate for 40s before blotting and air drying.
GridDetails: Copper 300 mesh continuous carbon grids (TAAB)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC
DateMar 23, 2010
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 10 e/Å2
Details: Data collected on a 4k x 4k CCD camera at 50000x magnification. Sampling interval was 1.76A/pixel.
Bits/pixel: 16

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Image processing

CTF correctionDetails: Each particle
Final angle assignmentDetails: Imagic
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, V, TIGRIS, EMAN / Number images used: 3680

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Atomic model buiding 1

Initial modelPDB ID:

4am6

SoftwareName: Chimera, CNS, Situs
DetailsProtocol: Rigid body. The Arp8 crystal structure was positioned interactively in Chimera, then the fit was refined using Situs and CNS.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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