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- EMDB-3084: Architecture and nucleotide-driven conformational states of the R... -

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Basic information

Entry
Database: EMDB / ID: EMD-3084
TitleArchitecture and nucleotide-driven conformational states of the Rvb1/Rvb2 dodecamer
Map dataATP-state Rvb1/2 focused reconstruction
Sample
  • Sample: ATP-state Rvb1/2 focused reconstruction
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2
KeywordsRvb1 / Rvb2
Function / homology
Function and homology information


R2TP complex / Swr1 complex / Ino80 complex / 5'-3' DNA helicase activity / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / DNA helicase activity / rRNA processing / DNA helicase ...R2TP complex / Swr1 complex / Ino80 complex / 5'-3' DNA helicase activity / box C/D snoRNP assembly / 3'-5' DNA helicase activity / NuA4 histone acetyltransferase complex / DNA helicase activity / rRNA processing / DNA helicase / protein stabilization / chromatin remodeling / DNA repair / chromatin / regulation of DNA-templated transcription / 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 / cryo EM / Resolution: 8.3 Å
AuthorsEwens CA / Su M / Zhao L / Houry WA / Southworth DR
CitationJournal: Structure / Year: 2016
Title: Architecture and Nucleotide-Dependent Conformational Changes of the Rvb1-Rvb2 AAA+ Complex Revealed by Cryoelectron Microscopy.
Authors: Caroline A Ewens / Min Su / Liang Zhao / Nardin Nano / Walid A Houry / Daniel R Southworth /
Abstract: Rvb1 and Rvb2 are essential AAA+ proteins that interact together during the assembly and activity of diverse macromolecules including chromatin remodelers INO80 and SWR-C, and ribonucleoprotein ...Rvb1 and Rvb2 are essential AAA+ proteins that interact together during the assembly and activity of diverse macromolecules including chromatin remodelers INO80 and SWR-C, and ribonucleoprotein complexes including telomerase and snoRNPs. ATP hydrolysis by Rvb1/2 is required for function; however, the mechanism that drives substrate remodeling is unknown. Here we determined the architecture of the yeast Rvb1/2 dodecamer using cryoelectron microscopy and identify that the substrate-binding insertion domain undergoes conformational changes in response to nucleotide state. 2D and 3D classification defines the dodecamer flexibility, revealing distinct arrangements and the hexamer-hexamer interaction interface. Reconstructions of the apo, ATP, and ADP states identify that Rvb1/2 undergoes substantial conformational changes that include a twist in the insertion-domain position and a corresponding rotation of the AAA+ ring. These results reveal how the ATP hydrolysis cycle of the AAA+ domains directs insertion-domain movements that could provide mechanical force during remodeling or helicase activities.
History
DepositionJul 8, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseJun 1, 2016-
UpdateJun 1, 2016-
Current statusJun 1, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3084.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationATP-state Rvb1/2 focused reconstruction
Voxel sizeX=Y=Z: 2 Å
Density
Contour LevelBy AUTHOR: 0.0262 / Movie #1: 0.0262
Minimum - Maximum-0.01926399 - 0.07261631
Average (Standard dev.)0.00012382 (±0.00466246)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0190.0730.000

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Supplemental data

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Sample components

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Entire : ATP-state Rvb1/2 focused reconstruction

EntireName: ATP-state Rvb1/2 focused reconstruction
Components
  • Sample: ATP-state Rvb1/2 focused reconstruction
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2

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Supramolecule #1000: ATP-state Rvb1/2 focused reconstruction

SupramoleculeName: ATP-state Rvb1/2 focused reconstruction / type: sample / ID: 1000 / Oligomeric state: heterododecamer / Number unique components: 2
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa
Method: size exclusion chromatography with multiangle light scattering

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Macromolecule #1: Rvb1

MacromoleculeName: Rvb1 / type: protein_or_peptide / ID: 1 / Name.synonym: Pontin / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pColaDuet
SequenceUniProtKB: RuvB-like protein 1

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Macromolecule #2: Rvb2

MacromoleculeName: Rvb2 / type: protein_or_peptide / ID: 2 / Name.synonym: Reptin / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pColaDuet
SequenceUniProtKB: RuvB-like protein 2

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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

Concentration0.3 mg/mL
BufferpH: 7.4
Details: 25mM HEPES, 150mM KCl, 6mM betaME, 5mM MgCl2, 200microM AMP-PNP
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV / Method: 1s blot

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateApr 23, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 1600 / Average electron dose: 5 e/Å2
Details: Every image is the average of 30 frames recorded by the direct electron detector
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: OTHER / Software - Name: relion, boxer, eman2, cftfind, spider / Number images used: 13630

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Atomic model buiding 1

Initial modelPDB ID:

Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: chimera, situs
DetailsThe domains were separately fitted by manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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