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- EMDB-1279: Different quaternary structures of human RECQ1 are associated wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-1279
TitleDifferent quaternary structures of human RECQ1 are associated with its dual enzymatic activity.
Map dataMap of K119R mutant RECQ1 bound to ssDNA, filtered using a 3D Gaussianlow-pass filter to a half-width of 17 Angstrom
Sample
  • Sample: K119R mutant RECQ1 and ssDNA
  • Protein or peptide: RECQ1
  • DNA: Single stranded DNA - oligo-dT-30
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 24.0 Å
AuthorsMuzzolini L / Beuron F / Patwardhan A / Popuri V / Cui S / Niccolini B / Rappas M / Freemont PS / Vindigni A
CitationJournal: PLoS Biol / Year: 2007
Title: Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity.
Authors: Laura Muzzolini / Fabienne Beuron / Ardan Patwardhan / Venkateswarlu Popuri / Sheng Cui / Benedetta Niccolini / Mathieu Rappas / Paul S Freemont / Alessandro Vindigni /
Abstract: RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual ...RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPgammaS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 A x 130 A with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.
History
DepositionOct 19, 2006-
Header (metadata) releaseOct 19, 2006-
Map releaseOct 19, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08503993
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.08503993
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1279.gif

Downloads & links

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Map

FileDownload / File: emd_1279.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of K119R mutant RECQ1 bound to ssDNA, filtered using a 3D Gaussianlow-pass filter to a half-width of 17 Angstrom
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.54 Å/pix.
x 100 pix.
= 254. Å		2.54 Å/pix.
x 100 pix.
= 254. Å		2.54 Å/pix.
x 100 pix.
= 254. 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 Level1: 0.0415 / Movie #1: 0.0850399
Minimum - Maximum-0.226079 - 0.341948
Average (Standard dev.)0.000989976 (±0.0269953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 254 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z254.000254.000254.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.2260.3420.001

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Supplemental data

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Sample components

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Entire : K119R mutant RECQ1 and ssDNA

EntireName: K119R mutant RECQ1 and ssDNA
Components
  • Sample: K119R mutant RECQ1 and ssDNA
  • Protein or peptide: RECQ1
  • DNA: Single stranded DNA - oligo-dT-30

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Supramolecule #1000: K119R mutant RECQ1 and ssDNA

SupramoleculeName: K119R mutant RECQ1 and ssDNA / type: sample / ID: 1000 / Oligomeric state: Probably hexameric / Number unique components: 2
Molecular weightExperimental: 400 KDa / Theoretical: 440 KDa / Method: Size exclusion chromatography

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Macromolecule #1: RECQ1

MacromoleculeName: RECQ1 / type: protein_or_peptide / ID: 1 / Name.synonym: RECQ1 / Number of copies: 6 / Oligomeric state: Possibly hexameric / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 400 KDa / Theoretical: 440 KDa

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Macromolecule #2: Single stranded DNA - oligo-dT-30

MacromoleculeName: Single stranded DNA - oligo-dT-30 / type: dna / ID: 2 / Name.synonym: ssDNA / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 7.4 / Details: 20mM TrisHCl, 150 mM KCl, 2 mM MgCl2
StainingType: NEGATIVE
Details: RECQ1 was incubated with a 1.5 molar excess of ssDNA, adsorbed onto a glow-discharged carbon-coated grid and negatively-stained with 1% uranyl acetate
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
DateJun 6, 2005
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 6.35 µm / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 48600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic / Number images used: 1163
Final angle assignmentDetails: Imagic
Final two d classificationNumber classes: 87

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