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- EMDB-14394: C. thermophilum Ku70/80 heterodimer bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-14394
TitleC. thermophilum Ku70/80 heterodimer bound to DNA
Map data
Sample
  • Complex: Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA
Biological speciesChaetomium ther (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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_14394.png

Downloads & links

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Map

FileDownload / File: emd_14394.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 338.88 Å		1.06 Å/pix.
x 320 pix.
= 338.88 Å		1.06 Å/pix.
x 320 pix.
= 338.88 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.32
Minimum - Maximum-9.623539 - 17.955511
Average (Standard dev.)0.009711243 (±0.2635293)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 338.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14394_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_14394_half_map_1.map
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Half map: #2

Fileemd_14394_half_map_2.map
Projections & Slices
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Sample components

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Entire : Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA

EntireName: Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA
Components
  • Complex: Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA

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Supramolecule #1: Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA

SupramoleculeName: Heterodimer of C. thermophilum Ku70/80 bound to double-stranded DNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Chaetomium ther (E. coli)
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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 43.19 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 211512
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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