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- EMDB-6218: Yeast Rvb1 and Rvb2 proteins oligomerize as a conformationally va... -

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Basic information

Entry
Database: EMDB / ID: EMD-6218
TitleYeast Rvb1 and Rvb2 proteins oligomerize as a conformationally variable dodecamer with low frequency
Map dataIn vivo assembled yeast Rvb1/Rvb2 complex (dodecamer, CL4)
Sample
  • Sample: In vivo assembled Rvb1/Rvb2 complex, CL4
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2
KeywordsAAA+ / Electron microscopy / Pontin / Reptin / Chromatin remodeling
Function / homology
Function and homology information


R2TP complex / Swr1 complex / Ino80 complex / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / DNA helicase activity / rRNA processing / 5'-3' DNA helicase activity / DNA helicase ...R2TP complex / Swr1 complex / Ino80 complex / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / DNA helicase activity / rRNA processing / 5'-3' DNA helicase activity / DNA helicase / protein stabilization / chromatin remodeling / DNA repair / regulation of DNA-templated transcription / chromatin / regulation of transcription by RNA polymerase II / ATP hydrolysis activity / ATP binding / nucleus
Similarity search - Function
RuvB-like / RuvB-like, AAA-lid domain / RuvBL1/2, DNA/RNA binding domain / TIP49 P-loop domain / TIP49 AAA-lid domain / TIP49, P-loop domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RuvB-like protein 1 / RuvB-like protein 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsJeganathan A / Leong A / Huen J / Zhao L / Nano N / Houry WA / Ortega J
CitationJournal: J Mol Biol / Year: 2015
Title: Yeast rvb1 and rvb2 proteins oligomerize as a conformationally variable dodecamer with low frequency.
Authors: Ajitha Jeganathan / Vivian Leong / Liang Zhao / Jennifer Huen / Nardin Nano / Walid A Houry / Joaquin Ortega /
Abstract: Rvb1 and Rvb2 are conserved AAA+ (ATPases associated with diverse cellular activities) proteins found at the core of large multicomponent complexes that play key roles in chromatin remodeling, ...Rvb1 and Rvb2 are conserved AAA+ (ATPases associated with diverse cellular activities) proteins found at the core of large multicomponent complexes that play key roles in chromatin remodeling, integrity of the telomeres, ribonucleoprotein complex biogenesis and other essential cellular processes. These proteins contain an AAA+ domain for ATP binding and hydrolysis and an insertion domain proposed to bind DNA/RNA. Yeast Rvb1 and Rvb2 proteins oligomerize primarily as heterohexameric rings. The six AAA+ core domains form the body of the ring and the insertion domains protrude from one face of the ring. Conversely, human Rvbs form a mixture of hexamers and dodecamers made of two stacked hexamers interacting through the insertion domains. Human dodecamers adopt either a contracted or a stretched conformation. Here, we found that yeast Rvb1/Rvb2 complexes when assembled in vivo mainly form hexamers but they also assemble as dodecamers with a frequency lower than 10%. Yeast dodecamers adopt not only the stretched and contracted structures that have been described for human Rvb1/Rvb2 dodecamers but also intermediate conformations in between these two extreme states. The orientation of the insertion domains of Rvb1 and Rvb2 proteins in these conformers changes as the dodecamer transitions from the stretched structure to a more contracted structure. Finally, we observed that for the yeast proteins, oligomerization as a dodecamer inhibits the ATPase activity of the Rvb1/Rvb2 complex. These results indicate that although human and yeast Rvb1 and Rvb2 proteins share high degree of homology, there are significant differences in their oligomeric behavior and dynamics.
History
DepositionDec 15, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 16, 2015-
UpdateFeb 10, 2016-
Current statusFeb 10, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6218.png

Downloads & links

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Map

FileDownload / File: emd_6218.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn vivo assembled yeast Rvb1/Rvb2 complex (dodecamer, CL4)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 120 pix.
= 304.8 Å		2.54 Å/pix.
x 120 pix.
= 304.8 Å		2.54 Å/pix.
x 120 pix.
= 304.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.54 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.03619136 - 0.0853504
Average (Standard dev.)-0.0005509 (±0.00898988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 304.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z304.800304.800304.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0360.085-0.001

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Supplemental data

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Sample components

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Entire : In vivo assembled Rvb1/Rvb2 complex, CL4

EntireName: In vivo assembled Rvb1/Rvb2 complex, CL4
Components
  • Sample: In vivo assembled Rvb1/Rvb2 complex, CL4
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2

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Supramolecule #1000: In vivo assembled Rvb1/Rvb2 complex, CL4

SupramoleculeName: In vivo assembled Rvb1/Rvb2 complex, CL4 / type: sample / ID: 1000
Oligomeric state: 6 molecules of Rvb1 and 6 molecules of Rvb2
Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa / Method: Size exclusion chromatography

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Macromolecule #1: Rvb1

MacromoleculeName: Rvb1 / type: protein_or_peptide / ID: 1
Name.synonym: RuvBL1, Tip49a, Tip48 / Tip49, ECP-54, Pontin, Tih1, p50, Tap54b
Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightExperimental: 50.5 KDa / Theoretical: 50.5 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) pRIL / Recombinant plasmid: pCOLADuet-1
SequenceUniProtKB: RuvB-like protein 1

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Macromolecule #2: Rvb2

MacromoleculeName: Rvb2 / type: protein_or_peptide / ID: 2
Name.synonym: RuvBL2, Tip49b, Tip49 / Tip48, ECP-51, Reptin, Tih2, p47, Tap54a
Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightExperimental: 51.6 KDa / Theoretical: 51.6 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) pRIL / Recombinant plasmid: pCOLADuet-1
SequenceUniProtKB: RuvB-like protein 2

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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: 25 mM Tris-HCl, pH 7.5, 80 mM KCl, 10% v/v glycerol, 1 mM DTT
StainingType: NEGATIVE / Details: 2% uranyl acetate
GridDetails: 400 mesh grids with thin carbon support, glow-discharged in air atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2010F
DateFeb 1, 2014
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: 100 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: JEOL

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Image processing

DetailsParticles selected with boxer, image classification done using ML3D, and projection matching 3D reconstruction done with Xmipp.
CTF correctionDetails: CTFFIND3
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: Xmipp / Number images used: 1718

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