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- EMDB-14393: Hairpin-bound state of the E. coli Mre11-Rad50 (SbcCD) head compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-14393
TitleHairpin-bound state of the E. coli Mre11-Rad50 (SbcCD) head complex bound to ADP and a DNA hairpin
Map data
Sample
  • Complex: Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin
    • Complex: Nuclease SbcCD
      • Protein or peptide: Nuclease SbcCD subunit D
      • Protein or peptide: Nuclease SbcCD subunit C
    • Complex: DNA hairpin
      • DNA: DNA hairpin (59-MER)
  • Ligand: MANGANESE (II) ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsABC-type ATPase / Nuclease / DNA repair / DNA BINDING PROTEIN
Function / homology
Function and homology information


double-stranded DNA endonuclease activity / DNA replication termination / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / 3'-5' exonuclease activity / double-strand break repair / DNA recombination / DNA replication ...double-stranded DNA endonuclease activity / DNA replication termination / DNA exonuclease activity / single-stranded DNA endodeoxyribonuclease activity / 3'-5'-DNA exonuclease activity / DNA repair complex / 3'-5' exonuclease activity / double-strand break repair / 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 / : / Mre11 nuclease, N-terminal metallophosphatase domain / SbcC/RAD50-like, Walker B motif / Rad50/SbcC-type AAA domain / AAA domain / Calcineurin-like phosphoesterase domain, ApaH type ...Nuclease SbcC, gammaproteobacteria type / Nuclease SbcCD subunit D, C-terminal domain / Type 5 capsule protein repressor C-terminal domain / Nuclease SbcCD subunit D / : / Mre11 nuclease, N-terminal metallophosphatase domain / SbcC/RAD50-like, Walker B motif / Rad50/SbcC-type AAA domain / AAA 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 D / Nuclease SbcCD subunit C
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / DNA molecule (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsGut F / Kaeshammer L
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-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14393.png

Downloads & links

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Map

FileDownload / File: emd_14393.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
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: 0.0198
Minimum - Maximum-0.049526863 - 0.09062077
Average (Standard dev.)0.00012215214 (±0.0016752583)
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_14393_msk_1.map
Projections & Slices
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Additional map: LAFTER-filtered map

Fileemd_14393_additional_1.map
AnnotationLAFTER-filtered map
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Half map: #2

Fileemd_14393_half_map_1.map
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Half map: #1

Fileemd_14393_half_map_2.map
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Sample components

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Entire : Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin

EntireName: Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin
Components
  • Complex: Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin
    • Complex: Nuclease SbcCD
      • Protein or peptide: Nuclease SbcCD subunit D
      • Protein or peptide: Nuclease SbcCD subunit C
    • Complex: DNA hairpin
      • DNA: DNA hairpin (59-MER)
  • Ligand: MANGANESE (II) ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin

SupramoleculeName: Complex of Mre11 dimer, Rad50 dimer and a DNA hairpin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Nuclease SbcCD

SupramoleculeName: Nuclease SbcCD / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: DNA hairpin

SupramoleculeName: DNA hairpin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: DNA molecule (others)

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Macromolecule #1: DNA hairpin (59-MER)

MacromoleculeName: DNA hairpin (59-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.144822 KDa
SequenceString: (DC)(DG)(DC)(DT)(DT)(DT)(DA)(DT)(DC)(DA) (DG)(DA)(DA)(DG)(DC)(DC)(DA)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DC)(DG)(DC)(DT) (DT)(DC)(DT)(DG)(DG)(DA)(DT)(DA)(DG)(DC) (DG) (DT)(DC)(DC)(DA)(DG)(DA) ...String:
(DC)(DG)(DC)(DT)(DT)(DT)(DA)(DT)(DC)(DA) (DG)(DA)(DA)(DG)(DC)(DC)(DA)(DG)(DA)(DC) (DA)(DT)(DT)(DA)(DA)(DC)(DG)(DC)(DT) (DT)(DC)(DT)(DG)(DG)(DA)(DT)(DA)(DG)(DC) (DG) (DT)(DC)(DC)(DA)(DG)(DA)(DA)(DG) (DC)(DG)(DT)(DT)(DA)(DA)(DT)(DG)(DT)(DC) (DT)(DG) (DG)(DC)(DT)(DT)(DC)(DT)(DG) (DA)(DT)(DA)(DA)(DA)(DG)(DC)(DG)

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Macromolecule #2: Nuclease SbcCD subunit D

MacromoleculeName: Nuclease SbcCD subunit D / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 45.640277 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRILHTSDWH LGQNFYSKSR EAEHQAFLDW LLETAQTHQV DAIIVAGDVF DTGSPPSYAR TLYNRFVVNL QQTGCHLVVL AGNQDSVAT LNESRDIMAF LNTTVVASAG HAPQILPRRD GTPGAVLCPI PFLRPRDIIT SQAGLNGIEK QQHLLAAITD Y YQQHYADA ...String:
MRILHTSDWH LGQNFYSKSR EAEHQAFLDW LLETAQTHQV DAIIVAGDVF DTGSPPSYAR TLYNRFVVNL QQTGCHLVVL AGNQDSVAT LNESRDIMAF LNTTVVASAG HAPQILPRRD GTPGAVLCPI PFLRPRDIIT SQAGLNGIEK QQHLLAAITD Y YQQHYADA CKLRGDQPLP IIATGHLTTV GASKSDAVRD IYIGTLDAFP AQNFPPADYI ALGHIHRAQI IGGMEHVRYC GS PIPLSFD ECGKSKYVHL VTFSNGKLES VENLNVPVTQ PMAVLKGDLA SITAQLEQWR DVSQEPPVWL DIEITTDEYL HDI QRKIQA LTESLPVEVL LVRRSREQRE RVLASQQRET LSELSVEEVF NRRLALEELD ESQQQRLQHL FTTTLHTLAG EHEA GHHHH HH

UniProtKB: Nuclease SbcCD subunit D

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Macromolecule #3: Nuclease SbcCD subunit C

MacromoleculeName: Nuclease SbcCD subunit C / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 118.851508 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKILSLRLKN LNSLKGEWKI DFTREPFASN GLFAITGPTG AGKTTLLDAI CLALYHETPR LSNVSQSQND LMTRDTAECL AEVEFEVKG EAYRAFWSQN RARNQPDGNL QVPRVELARC ADGKILADKV KDKLELTATL TGLDYGRFTR SMLLSQGQFA A FLNAKPKE ...String:
MKILSLRLKN LNSLKGEWKI DFTREPFASN GLFAITGPTG AGKTTLLDAI CLALYHETPR LSNVSQSQND LMTRDTAECL AEVEFEVKG EAYRAFWSQN RARNQPDGNL QVPRVELARC ADGKILADKV KDKLELTATL TGLDYGRFTR SMLLSQGQFA A FLNAKPKE RAELLEELTG TEIYGQISAM VFEQHKSART ELEKLQAQAS GVTLLTPEQV QSLTASLQVL TDEEKQLITA QQ QEQQSLN WLTRQDELQQ EASRRQQALQ QALAEEEKAQ PQLAALSLAQ PARNLRPHWE RIAEHSAALA HIRQQIEEVN TRL QSTMAL RASIRHHAAK QSAELQQQQQ SLNTWLQEHD RFRQWNNEPA GWRAQFSQQT SDREHLRQWQ QQLTHAEQKL NALA AITLT LTADEVATAL AQHAEQRPLR QHLVALHGQI VPQQKRLAQL QVAIQNVTQE QTQRNAALNE MRQRYKEKTQ QLADV KTIC EQEARIKTLE AQRAQLQAGQ PCPLCGSTSH PAVEAYQALE PGVNQSRLLA LENEVKKLGE EGATLRGQLD AITKQL QRD ENEAQSLRQD EQALTQQWQA VTASLNITLQ PLDDIQPWLD AQDEHERQLR LLSQRHELQG QIAAHNQQII QYQQQIE QR QQLLLTTLTG YALTLPQEDE EESWLATRQQ EAQSWQQRQN ELTALQNRIQ QLTPILETLP QSDELPHCEE TVVLENWR Q VHEQCLALHS QQQTLQQQDV LAAQSLQKAQ AQFDTALQAS VFDDQQAFLA ALMDEQTLTQ LEQLKQNLEN QRRQAQTLV TQTAETLAQH QQHRPDDGLA LTVTVEQIQQ ELAQTHQKLR ENTTSQGEIR QQLKQDADNR QQQQTLMQQI AQMTQQVEDW GYLNSLIGS KEGDKFRKFA QGLTLDNLVH LANQQLTRLH GRYLLQRKAS EALEVEVVDT WQADAVRDTR TLSGGESFLV S LALALALS DLVSHKTRID SLFLDEGFGT LDSETLDTAL DALDALNASG KTIGVISHVE AMKERIPVQI KVKKINGLGY SK LESTFAV K

UniProtKB: Nuclease SbcCD subunit C

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Macromolecule #4: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 46.8 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

Particle selectionNumber selected: 54504
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6s85

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61845
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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