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- EMDB-10114: The E.coli Mre11-Rad50 complex (SbcCD) bound to ADP and dsDNA wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-10114
TitleThe E.coli Mre11-Rad50 complex (SbcCD) bound to ADP and dsDNA with the first coiled-coil interaction site
Map data
Sample
  • Complex: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
Function / homology
Function and homology information


double-stranded DNA endonuclease activity / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / exonuclease activity / ATP-dependent DNA damage sensor activity / 3'-5' exonuclease activity / double-strand break repair / endonuclease activity ...double-stranded DNA endonuclease activity / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / exonuclease activity / ATP-dependent DNA damage sensor activity / 3'-5' exonuclease activity / double-strand break repair / endonuclease activity / DNA recombination / DNA replication / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
Nuclease SbcC, gammaproteobacteria type / Nuclease SbcCD subunit D, C-terminal domain / Type 5 capsule protein repressor C-terminal domain / Nuclease SbcCD subunit D / SbcC/RAD50-like, Walker B motif / Rad50/SbcC-type AAA domain / AAA domain / Mre11 nuclease, N-terminal metallophosphatase domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase ...Nuclease SbcC, gammaproteobacteria type / Nuclease SbcCD subunit D, C-terminal domain / Type 5 capsule protein repressor C-terminal domain / Nuclease SbcCD subunit D / SbcC/RAD50-like, Walker B motif / Rad50/SbcC-type AAA domain / AAA domain / Mre11 nuclease, N-terminal metallophosphatase domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nuclease SbcCD subunit C / Nuclease SbcCD subunit D / Nuclease SbcCD subunit D / Nuclease SbcCD subunit C
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsKaeshammer L / Saathoff JH / Gut F / Bartho J / Alt A / Kessler B / Lammens K / Hopfner KP
Funding support Germany, 2 items
OrganizationGrant numberCountry
European Research Council
German Research Foundation Germany
CitationJournal: Mol Cell / Year: 2019
Title: Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.
Authors: Lisa Käshammer / Jan-Hinnerk Saathoff / Katja Lammens / Fabian Gut / Joseph Bartho / Aaron Alt / Brigitte Kessler / Karl-Peter Hopfner /
Abstract: DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11- ...DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.
History
DepositionJul 8, 2019-
Header (metadata) releaseSep 4, 2019-
Map releaseSep 4, 2019-
UpdateNov 20, 2019-
Current statusNov 20, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0091
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0091
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10114.png

Downloads & links

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Map

FileDownload / File: emd_10114.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.0091 / Movie #1: 0.0091
Minimum - Maximum-0.017892994 - 0.043078568
Average (Standard dev.)0.00006128934 (±0.001980673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z246.000246.000246.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0180.0430.000

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Supplemental data

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Sample components

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Entire : Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in co...

EntireName: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
Components
  • Complex: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA

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Supramolecule #1: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in co...

SupramoleculeName: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 12811 / Average electron dose: 73.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Details: The initial model was calculated de novo using cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 152271

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