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- EMDB-2164: Dodecameric human RuvBL1-RuvBL2 complex (stretched conformation) -

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Basic information

Entry
Database: EMDB / ID: EMD-2164
TitleDodecameric human RuvBL1-RuvBL2 complex (stretched conformation)
Map dataReconstruction of the RuvBL1-RuvBL2 complex (stretched conformation)
Sample
  • Sample: Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conformation)
  • Protein or peptide: RuvBL1RuvB-like 1
  • Protein or peptide: RuvBL2
KeywordsRuvBL1 / RuvBL2 / Rvb1 / Rvb2 / Pontin / Reptin / AAA+
Function / homologyPapC-like, C-terminal domain / NuA4 histone acetyltransferase complex
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsLopez-Perrote A / Munoz-Hernandez H / Gil D / Llorca O
CitationJournal: Nucleic Acids Res / Year: 2012
Title: Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1-RuvBL2 complex.
Authors: Andrés López-Perrote / Hugo Muñoz-Hernández / David Gil / Oscar Llorca /
Abstract: RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and ...RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1-RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1-RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1-RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.
History
DepositionJul 26, 2012-
Header (metadata) releaseSep 19, 2012-
Map releaseSep 19, 2012-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2164.jpg

Downloads & links

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Map

FileDownload / File: emd_2164.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the RuvBL1-RuvBL2 complex (stretched conformation)
Voxel sizeX=Y=Z: 3.45 Å
Density
Contour LevelBy AUTHOR: 3.8 / Movie #1: 3.8
Minimum - Maximum-6.2162962 - 9.335557939999999
Average (Standard dev.)-0.00186431 (±0.97464311)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions104104104
Spacing104104104
CellA=B=C: 358.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.453.453.45
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z358.800358.800358.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS104104104
D min/max/mean-6.2169.336-0.002

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Supplemental data

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Sample components

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Entire : Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conf...

EntireName: Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conformation)
Components
  • Sample: Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conformation)
  • Protein or peptide: RuvBL1RuvB-like 1
  • Protein or peptide: RuvBL2

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Supramolecule #1000: Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conf...

SupramoleculeName: Reconstruction of the human RuvBL1-RuvBL2 complex (stretched conformation)
type: sample / ID: 1000 / Oligomeric state: Dodecameric / Number unique components: 2
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: RuvBL1

MacromoleculeName: RuvBL1 / type: protein_or_peptide / ID: 1 / Name.synonym: Rvb1, Pontin, TIP49, TIP49a / Details: full length / Number of copies: 6 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET15b
SequenceGO: NuA4 histone acetyltransferase complex / InterPro: PapC-like, C-terminal domain

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Macromolecule #2: RuvBL2

MacromoleculeName: RuvBL2 / type: protein_or_peptide / ID: 2 / Name.synonym: Rvb2, Reptin, TIP48, TIP49b / Details: full length / Number of copies: 6 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pCDFDuet-1
SequenceGO: NuA4 histone acetyltransferase complex

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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

Concentration0.25 mg/mL
BufferpH: 7.4 / Details: 250mM NaCl, 25 mM Tris-HCL
GridDetails: Quantifoil grids 300 mesh R2/1 holey carbon copper grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
Method: Manual application (4 microliters) Blot offset: -2 mm Blot total:2 Blot time: 2s Wait time: 30s Drain time: 1s

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 86855 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: In-column nergy filter (Omega Filter) / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 10.0 eV
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 83 K / Max: 100 K / Average: 91.5 K
Alignment procedureLegacy - Astigmatism: Phase flipping (Objective lens astigmatism was corrected using the CCD and the power spectrum)
DateMar 28, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 338 / Average electron dose: 12 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each CCD Frame, estimated with CTFFIND and corrected using BSOFT
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1.9, EMAN2, XMIPP, BSOFT / Number images used: 6615

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Atomic model buiding 1

Initial modelPDB ID:

2c9o

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation

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Atomic model buiding 2

Initial modelPDB ID:

2xsz

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation

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