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- EMDB-10116: Cutting state of the E. coli Mre11-Rad50 (SbcCD) head complex bou... -

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Basic information

Entry
Database: EMDB / ID: EMD-10116
TitleCutting state of the E. coli Mre11-Rad50 (SbcCD) head complex bound to ADP and dsDNA.
Map data
Sample
  • Complex: Heterotetrameric complex of full-length Mre11-Rad50 (SbcCD) in complex with ADP and dsDNA
    • Complex: Mre11-Rad50
      • Protein or peptide: Nuclease SbcCD subunit C
      • Protein or peptide: Nuclease SbcCD subunit D
    • Complex: dsDNA
      • DNA: DNA (31-MER)
      • DNA: DNA (32-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION
KeywordsNuclease / DNA repair / ABC-type ATPase / DNA double-strand breaks / DNA BINDING PROTEIN
Function / homology
Function and homology information


DNA exonuclease activity / double-stranded DNA endonuclease activity / single-stranded DNA endodeoxyribonuclease activity / DNA repair complex / exonuclease activity / 3'-5'-DNA exonuclease activity / 3'-5' exonuclease activity / double-strand break repair / endonuclease activity / DNA recombination ...DNA exonuclease activity / double-stranded DNA endonuclease activity / single-stranded DNA endodeoxyribonuclease activity / DNA repair complex / exonuclease activity / 3'-5'-DNA exonuclease 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 / Mre11 nuclease, N-terminal metallophosphatase domain / Rad50/SbcC-type AAA domain / AAA 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 / Mre11 nuclease, N-terminal metallophosphatase domain / 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 C / Nuclease SbcCD subunit D / Nuclease SbcCD subunit D / Nuclease SbcCD subunit C
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKaeshammer L / Saathoff JH
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) releaseJul 24, 2019-
Map releaseSep 4, 2019-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s85
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10116.png

Downloads & links

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Map

FileDownload / File: emd_10116.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: 0.009 / Movie #1: 0.009
Minimum - Maximum-0.028870141 - 0.055968318
Average (Standard dev.)0.00005468869 (±0.0021747735)
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-0.0290.0560.000

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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
    • Complex: Mre11-Rad50
      • Protein or peptide: Nuclease SbcCD subunit C
      • Protein or peptide: Nuclease SbcCD subunit D
    • Complex: dsDNA
      • DNA: DNA (31-MER)
      • DNA: DNA (32-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION

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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

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Supramolecule #2: Mre11-Rad50

SupramoleculeName: Mre11-Rad50 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: dsDNA

SupramoleculeName: dsDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Nuclease SbcCD subunit C

MacromoleculeName: Nuclease SbcCD subunit C / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 118.851508 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 #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: 45.599227 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRILHTSDWH LGQNFYSKSR EAEHQAFLDW LLETAQTHQV DAIIVAGDVF DTGSPPSYAR TLYNRFVVNL QQTGCHLVVL AGNSDSVAT LNESRDIMAF LNTTVVASAG HAPQILPRRD GTPGAVLCPI PFLRPRDIIT SQAGLNGIEK QQHLLAAITD Y YQQHYADA ...String:
MRILHTSDWH LGQNFYSKSR EAEHQAFLDW LLETAQTHQV DAIIVAGDVF DTGSPPSYAR TLYNRFVVNL QQTGCHLVVL AGNSDSVAT 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: DNA (31-MER)

MacromoleculeName: DNA (31-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.518891 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)(DG)(DA)(DA)(DA) (DC) (DT)(DC)(DA)(DA)(DC)(DG) ...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)(DG)(DA)(DA)(DA) (DC) (DT)(DC)(DA)(DA)(DC)(DG)(DA)(DG) (DC)(DT)(DG)(DG)(DA)(DC)(DG)(DC)(DG)(DG) (DA)(DT)

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Macromolecule #4: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.464807 KDa
SequenceString: (DA)(DT)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DC) (DA)(DG)(DC)(DT)(DC)(DG)(DT)(DT)(DG)(DA) (DG)(DT)(DT)(DT)(DC)(DT)(DC)(DC)(DA) (DG)(DA)(DA)(DG)(DC)(DG)(DT)(DT)(DA)(DA) (DT) (DG)(DT)(DC)(DT)(DG)(DG) ...String:
(DA)(DT)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DC) (DA)(DG)(DC)(DT)(DC)(DG)(DT)(DT)(DG)(DA) (DG)(DT)(DT)(DT)(DC)(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 #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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Macromolecule #7: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: MN
Molecular weightTheoretical: 54.938 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
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 152271
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
PDB IDChain

4m0v

chain_id: A, source_name: PDB, initial_model_type: experimental model

4m0v

chain_id: B, source_name: PDB, initial_model_type: experimental model
Output model

PDB-6s85:
Cutting state of the E. coli Mre11-Rad50 (SbcCD) head complex bound to ADP and dsDNA.

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