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- EMDB-5228: Alternative Oligomeric States of the Yeast Rvb1-Rvb2 Complex Indu... -

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Basic information

Entry
Database: EMDB / ID: EMD-5228
TitleAlternative Oligomeric States of the Yeast Rvb1-Rvb2 Complex Induced by Histidine Tags
Map data3D Reconstruction of His-tagged Rvb1-Rvb2 complex
Sample
  • Sample: Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged Rvb2 proteins
  • Protein or peptide: helicase
KeywordsRvb1 / Rvb2
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsCheung KLY / Huen J / Kakihara Y / Houry WA / Ortega J
CitationJournal: J Mol Biol / Year: 2010
Title: Alternative oligomeric states of the yeast Rvb1/Rvb2 complex induced by histidine tags.
Authors: Kevin L Y Cheung / Jennifer Huen / Yoshito Kakihara / Walid A Houry / Joaquin Ortega /
Abstract: Rvb1 and Rvb2 are essential AAA(+) (ATPases associated with diverse cellular activities) helicases, which are important components of critical complexes such as chromatin remodeling and telomerase ...Rvb1 and Rvb2 are essential AAA(+) (ATPases associated with diverse cellular activities) helicases, which are important components of critical complexes such as chromatin remodeling and telomerase complexes. The oligomeric state of the Rvb proteins has been controversial. Independent studies from several groups have described the yeast and human Rvb1/Rvb2 complex both as a single and as a double hexameric ring complex. We found that histidine-tagged constructs of yeast Rvb proteins employed in some of these studies induced the assembly of double hexameric ring Rvb1/Rvb2 complexes. Instead, untagged versions of these proteins assemble into single hexameric rings. Furthermore, purified endogenous untagged Rvb1/Rvb2 complexes from Saccharomyces cerevisiae were also found as single hexameric rings, similar to the complexes assembled in vitro from the purified untagged components. These results demonstrate that some of the differences between the reported structures are caused by histidine tags and imply that further studies on the purified proteins should be carried out using untagged constructs.
History
DepositionAug 17, 2010-
Header (metadata) releaseAug 24, 2010-
Map releaseNov 17, 2010-
UpdateNov 17, 2010-
Current statusNov 17, 2010Processing 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 cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5228_1.tif

Downloads & links

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Map

FileDownload / File: emd_5228.map.gz / Format: CCP4 / Size: 5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Reconstruction of His-tagged Rvb1-Rvb2 complex
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.0295023 - 0.0798848
Average (Standard dev.)0.000267295 (±0.0101801)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-55-55-55
Dimensions110110110
Spacing110110110
CellA=B=C: 279.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z110110110
origin x/y/z0.0000.0000.000
length x/y/z279.400279.400279.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-55-55-55
NC/NR/NS110110110
D min/max/mean-0.0300.0800.000

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Supplemental data

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Sample components

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Entire : Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged R...

EntireName: Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged Rvb2 proteins
Components
  • Sample: Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged Rvb2 proteins
  • Protein or peptide: helicase

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Supramolecule #1000: Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged R...

SupramoleculeName: Rvb1-Rvb2 complex assembled with His-tagged Rvb1 and non-tagged Rvb2 proteins
type: sample / ID: 1000 / Oligomeric state: Dodecamer / Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa
Method: Size exclusion chromatography and Blue native polyacrylamide gel electrophoresis

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Macromolecule #1: helicase

MacromoleculeName: helicase / type: protein_or_peptide / ID: 1 / Name.synonym: Rvb1 and Rvb2 / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Saccharomyces cerevisiae
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pProEx (Rvb1) and p11 (Rvb2)

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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

BufferpH: 7.5 / Details: 25mM Tris, 80mM KCl, 10% glycerol
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl acetate for 1 minute
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureAverage: 298.15 K
Detailsimages were taken with FasTem low dose kit
DateApr 28, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 50 / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single-tilt / Specimen holder model: JEOL

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP / Number images used: 10000

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Atomic model buiding 1

Initial modelPDB ID:

2c9o

DetailsProtocol: Rigid body. AAA domains were docked separately from the insertion domain manually with Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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