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- EMDB-14392: Composite map of two E. coli Mre11-Rad50 (SbcCD) complexes bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-14392
TitleComposite map of two E. coli Mre11-Rad50 (SbcCD) complexes bound to Ku70/80 blocked dsDNA in endonuclease state
Map dataComposite map
Sample
  • Complex: Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 blocked double-stranded DNA
    • Complex: E. coli Mre11-Rad50 (SbcCD) bound to dsDNA
    • Complex: C. thermophilum Ku70/80 bound to dsDNA
Biological speciesEscherichia coli (E. coli) / Chaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsGut F / Kaeshammer L / Lammens K / Bartho J / van de Logt E / Kessler B / Hopfner KP
Funding supportEuropean Union, Germany, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
German Research Foundation (DFG) Germany
CitationJournal: Mol Cell / Year: 2022
Title: Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50.
Authors: Fabian Gut / Lisa Käshammer / Katja Lammens / Joseph D Bartho / Anna-Maria Boggusch / Erik van de Logt / Brigitte Kessler / Karl-Peter Hopfner /
Abstract: DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood ...DNA double-strand breaks (DSBs) threaten genome stability and are linked to tumorigenesis in humans. Repair of DSBs requires the removal of attached proteins and hairpins through a poorly understood but physiologically critical endonuclease activity by the Mre11-Rad50 complex. Here, we report cryoelectron microscopy (cryo-EM) structures of the bacterial Mre11-Rad50 homolog SbcCD bound to a protein-blocked DNA end and a DNA hairpin. The structures reveal that Mre11-Rad50 bends internal DNA for endonucleolytic cleavage and show how internal DNA, DNA ends, and hairpins are processed through a similar ATP-regulated conformational state. Furthermore, Mre11-Rad50 is loaded onto blocked DNA ends with Mre11 pointing away from the block, explaining the distinct biochemistries of 3' → 5' exonucleolytic and endonucleolytic incision through the way Mre11-Rad50 interacts with diverse DNA ends. In summary, our results unify Mre11-Rad50's enigmatic nuclease diversity within a single structural framework and reveal how blocked DNA ends and hairpins are processed.
History
DepositionFeb 21, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14392.png

Downloads & links

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Map

FileDownload / File: emd_14392.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 600 pix.
= 635.4 Å		1.06 Å/pix.
x 600 pix.
= 635.4 Å		1.06 Å/pix.
x 600 pix.
= 635.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 20.0
Minimum - Maximum-84.121445 - 165.06259
Average (Standard dev.)0.0035820112 (±1.9627719)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 635.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14392_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_14392_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14392_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14392_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 b...

EntireName: Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 blocked double-stranded DNA
Components
  • Complex: Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 blocked double-stranded DNA
    • Complex: E. coli Mre11-Rad50 (SbcCD) bound to dsDNA
    • Complex: C. thermophilum Ku70/80 bound to dsDNA

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Supramolecule #1: Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 b...

SupramoleculeName: Ku-RM-MR-Ku state of two Mre11-Rad50 complexes bound to Ku70/80 blocked double-stranded DNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #2: E. coli Mre11-Rad50 (SbcCD) bound to dsDNA

SupramoleculeName: E. coli Mre11-Rad50 (SbcCD) bound to dsDNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: C. thermophilum Ku70/80 bound to dsDNA

SupramoleculeName: C. thermophilum Ku70/80 bound to dsDNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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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

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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 / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 43.19 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 10494

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