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- EMDB-3243: Dodecameric Chaetomium thermophilum Rvb1/Rvb2 complex in stretche... -

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Basic information

Entry
Database: EMDB / ID: EMD-3243
TitleDodecameric Chaetomium thermophilum Rvb1/Rvb2 complex in stretched conformation
Map dataCryo-EM reconstruction of Chaetomium thermophilum dodecameric Rvb1/Rvb2 complex in stretched conformation
Sample
  • Sample: Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex in stretched conformation
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2
KeywordsAAA+ proteins / cryo-electron microscopy / Rvb1 / Rvb2 / Chaetomium thermophilum
Function / homology
Function and homology information


ATP-dependent activity, acting on DNA / helicase activity / chromatin organization / DNA helicase / DNA repair / ATP hydrolysis activity / ATP binding / nucleus
Similarity search - Function
RuvB-like / 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 / TIP49, P-loop domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain ...RuvB-like / 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 / TIP49, P-loop domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RuvB-like helicase / RuvB-like helicase
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 21.4 Å
AuthorsSilva-Martin N / Dauden MI / Glatt S / Hoffmann NA / Kastritis P / Bork P / Beck M / Mueller CW
CitationJournal: PLoS One / Year: 2016
Title: The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex.
Authors: Noella Silva-Martin / María I Daudén / Sebastian Glatt / Niklas A Hoffmann / Panagiotis Kastritis / Peer Bork / Martin Beck / Christoph W Müller /
Abstract: The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, ...The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.
History
DepositionNov 13, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseJan 20, 2016-
UpdateJan 20, 2016-
Current statusJan 20, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0573
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0573
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3243.tif

Downloads & links

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Map

FileDownload / File: emd_3243.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of Chaetomium thermophilum dodecameric Rvb1/Rvb2 complex in stretched conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.34 Å/pix.
x 74 pix.
= 321.16 Å		4.34 Å/pix.
x 74 pix.
= 321.16 Å		4.34 Å/pix.
x 74 pix.
= 321.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0573 / Movie #1: 0.0573
Minimum - Maximum-0.12715153 - 0.15276451
Average (Standard dev.)0.00011426 (±0.01710674)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions747474
Spacing747474
CellA=B=C: 321.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.344.344.34
M x/y/z747474
origin x/y/z0.0000.0000.000
length x/y/z321.160321.160321.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS747474
D min/max/mean-0.1270.1530.000

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Supplemental data

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Sample components

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Entire : Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex...

EntireName: Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex in stretched conformation
Components
  • Sample: Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex in stretched conformation
  • Protein or peptide: Rvb1
  • Protein or peptide: Rvb2

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Supramolecule #1000: Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex...

SupramoleculeName: Full-length Chaetomium thermophilum Rvb1/Rvb2 dodecameric complex in stretched conformation
type: sample / ID: 1000
Oligomeric state: Dodecamer formed by two hetero-hexamers of Rvb1/Rvb2
Number unique components: 2
Molecular weightTheoretical: 621 KDa

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Macromolecule #1: Rvb1

MacromoleculeName: Rvb1 / type: protein_or_peptide / ID: 1 / Name.synonym: RuvBL1, Pontin, TIP49a / Number of copies: 6 / Oligomeric state: Double hetero-hexamer with Rvb2 / Recombinant expression: Yes
Source (natural)Organism: Chaetomium thermophilum (fungus) / synonym: thermophilic filamentous fungus
Molecular weightTheoretical: 50.384 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) Star pRARE strain / Recombinant plasmid: pET-MCN
SequenceUniProtKB: RuvB-like helicase
InterPro: AAA+ ATPase domain, P-loop containing nucleoside triphosphate hydrolase, RuvB-like, TIP49, P-loop domain

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Macromolecule #2: Rvb2

MacromoleculeName: Rvb2 / type: protein_or_peptide / ID: 2 / Name.synonym: RuvBL2, Reptin, TIP49b / Number of copies: 6 / Oligomeric state: Double hetero-hexamer with Rvb2 / Recombinant expression: Yes
Source (natural)Organism: Chaetomium thermophilum (fungus) / synonym: thermophilic filamentous fungus
Molecular weightTheoretical: 53.1458 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) Star pRARE strain / Recombinant plasmid: pET-MCN
SequenceUniProtKB: RuvB-like helicase
InterPro: AAA+ ATPase domain, P-loop containing nucleoside triphosphate hydrolase, RuvB-like, TIP49, P-loop domain

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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

Concentration0.35 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCl pH 7.5, 125 mM NaCl, 2 mM 2-mercaptoethanol and 5 mM MgCl2
GridDetails: glow-discharged molybdenum grids 400 mesh, 1.2/1.5, Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III
Method: 9 seconds blotting time, 15 seconds waiting time, blot offset -3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateNov 3, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1629 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were binned 4.
CTF correctionDetails: CTFFIND3
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 21.4 Å / Resolution method: OTHER / Software - Name: RELION, EMAN, XMIPP, Spider / Number images used: 1810
Final two d classificationNumber classes: 200
FSC plot (resolution estimation)

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