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- EMDB-2808: Electron cryo-microscopy of the HerA-NurA double strand break res... -

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Basic information

Entry
Database: EMDB / ID: EMD-2808
TitleElectron cryo-microscopy of the HerA-NurA double strand break resection complex
Map dataReconstruction of the HerA-NurA double strand break resection complex from Sulfolobus solfataricus
Sample
  • Sample: HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus
  • Protein or peptide: HerA
  • Protein or peptide: NurA
KeywordsHelicase / Nuclease / FtsK/HerA ATPase / DNA double-strand break repair / DNA resection
Function / homology
Function and homology information


nuclease activity / DNA 5'-3' helicase / DNA 3'-5' helicase / helicase activity / Hydrolases; Acting on ester bonds / DNA repair / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Helicase HerA, barrel domain / : / : / HAS barrel domain / NurA domain / NurA domain / NurA / Helicase HerA-like / Helicase HerA, central domain / Helicase HerA, central domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA double-strand break repair helicase HerA / DNA double-strand break repair nuclease NurA
Similarity search - Component
Biological speciesSulfolobus solfataricus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.35 Å
AuthorsByrne RT / Schuller JM / Unverdorben P / Foerster F / Hopfner K-P
CitationJournal: FEBS Lett / Year: 2014
Title: Molecular architecture of the HerA-NurA DNA double-strand break resection complex.
Authors: Robert Thomas Byrne / Jan Michael Schuller / Pia Unverdorben / Friedrich Förster / Karl-Peter Hopfner /
Abstract: DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, ...DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.
History
DepositionOct 31, 2014-
Header (metadata) releaseNov 12, 2014-
Map releaseNov 19, 2014-
UpdateDec 24, 2014-
Current statusDec 24, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2808.tif

emd_2808.tif

Downloads & links

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Map

FileDownload / File: emd_2808.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the HerA-NurA double strand break resection complex from Sulfolobus solfataricus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 150 pix.
= 265.5 Å		1.77 Å/pix.
x 150 pix.
= 265.5 Å		1.77 Å/pix.
x 150 pix.
= 265.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.15550664 - 0.18389343
Average (Standard dev.)0.00030055 (±0.00911862)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 265.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z265.500265.500265.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.1560.1840.000

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Supplemental data

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Sample components

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Entire : HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus

EntireName: HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus
Components
  • Sample: HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus
  • Protein or peptide: HerA
  • Protein or peptide: NurA

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Supramolecule #1000: HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus

SupramoleculeName: HerA from Sulfolobus Solfataricus NurA from Sulfolobus Solfataricus
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: One homohexamer of HerA binds to one homodimer of NurA
Number unique components: 2
Molecular weightExperimental: 419 KDa / Theoretical: 416 KDa / Method: SEC-RALS

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Macromolecule #1: HerA

MacromoleculeName: HerA / type: protein_or_peptide / ID: 1 / Name.synonym: SSO2251 / Number of copies: 6 / Oligomeric state: homohexamer / Recombinant expression: Yes
Source (natural)Organism: Sulfolobus solfataricus (archaea) / Strain: P2
Molecular weightTheoretical: 56 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta / Recombinant plasmid: pET-Duet-1
SequenceUniProtKB: DNA double-strand break repair helicase HerA

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Macromolecule #2: NurA

MacromoleculeName: NurA / type: protein_or_peptide / ID: 2 / Name.synonym: SSO2248 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Sulfolobus solfataricus (archaea) / Strain: P2
Molecular weightTheoretical: 39 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta / Recombinant plasmid: pET-Duet-1
SequenceUniProtKB: DNA double-strand break repair nuclease NurA

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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

Concentration0.05 mg/mL
BufferpH: 8 / Details: 100 mM NaCl, 20 mM Hepes
GridDetails: 2:1 holey carbon grids (Quantifoil Micro Tools, Germany)
VitrificationCryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Method: Blot for 2 seconds before plunging, wash twice with water

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Specialist opticsEnergy filter - Name: GIF QUANTUM / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateAug 22, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 863 / Average electron dose: 25 e/Å2
Details: Every image is the average of 20 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
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 semi-manually selected in e2boxer and further processed in RELION
CTF correctionDetails: On the micrograph level
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.35 Å / Resolution method: OTHER / Software - Name: RELION / Details: see detailed method in paper / Number images used: 100000

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