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- EMDB-14391: Endonuclease state of the E. coli Mre11-Rad50 (SbcCD) head comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-14391
TitleEndonuclease state of the E. coli Mre11-Rad50 (SbcCD) head complex bound to ADP and dsDNA
Map data
Sample
  • Complex: Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA
    • Protein or peptide: Nuclease SbcCD subunit C
    • Protein or peptide: Nuclease SbcCD subunit D
    • DNA: DNA (31-MER)
    • DNA: DNA (31-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION
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 / 3'-5' exonuclease activity / double-strand break repair / DNA recombination / DNA replication / DNA repair ...double-stranded DNA endonuclease activity / 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 / 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 D / Nuclease SbcCD subunit C
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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_14391.png

Downloads & links

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Map

FileDownload / File: emd_14391.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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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-0.07378133 - 0.13127208
Average (Standard dev.)9.851988e-05 (±0.0020471576)
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_14391_msk_1.map
Projections & Slices
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Additional map: LAFTER-filtered cryo-EM map

Fileemd_14391_additional_1.map
AnnotationLAFTER-filtered cryo-EM map
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Half map: #2

Fileemd_14391_half_map_1.map
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Half map: #1

Fileemd_14391_half_map_2.map
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Sample components

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Entire : Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA

EntireName: Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA
Components
  • Complex: Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA
    • Protein or peptide: Nuclease SbcCD subunit C
    • Protein or peptide: Nuclease SbcCD subunit D
    • DNA: DNA (31-MER)
    • DNA: DNA (31-MER)
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA

SupramoleculeName: Complex of Mre11 dimer, Rad50 dimer and double-stranded DNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Nuclease SbcCD subunit C

MacromoleculeName: Nuclease SbcCD subunit C / type: protein_or_peptide / ID: 1
Details: A large part of the structure in the middle of the sequence is missing. Therefore the alignment is not correct. The C-terminal part misaligns to the middle domain of the sequence.
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

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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

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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: 37.099617 KDa
SequenceString: (DC)(DC)(DA)(DG)(DT)(DT)(DG)(DA)(DT)(DC) (DG)(DG)(DC)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC) (DG)(DC)(DG)(DA)(DC)(DA)(DA)(DT)(DT)(DT) (DG) (DC)(DG)(DA)(DC)(DG)(DG) ...String:
(DC)(DC)(DA)(DG)(DT)(DT)(DG)(DA)(DT)(DC) (DG)(DG)(DC)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DC)(DG)(DC)(DC) (DG)(DC)(DG)(DA)(DC)(DA)(DA)(DT)(DT)(DT) (DG) (DC)(DG)(DA)(DC)(DG)(DG)(DC)(DG) (DC)(DG)(DT)(DG)(DC)(DA)(DG)(DG)(DG)(DC) (DC)(DA) (DG)(DA)(DC)(DT)(DG)(DG)(DA) (DG)(DG)(DT)(DG)(DG)(DC)(DA)(DA)(DC)(DG) (DC)(DC)(DA) (DA)(DT)(DC)(DA)(DG)(DC) (DA)(DA)(DC)(DG)(DA)(DC)(DT)(DG)(DT)(DT) (DT)(DG)(DC)(DC) (DC)(DG)(DC)(DC)(DA) (DG)(DT)(DT)(DG)(DT)(DT)(DG)(DT)(DG)(DC) (DC)(DA)(DC)(DG)(DC)

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Macromolecule #4: DNA (31-MER)

MacromoleculeName: DNA (31-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 36.970527 KDa
SequenceString: (DG)(DC)(DG)(DT)(DG)(DG)(DC)(DA)(DC)(DA) (DA)(DC)(DA)(DA)(DC)(DT)(DG)(DG)(DC)(DG) (DG)(DG)(DC)(DA)(DA)(DA)(DC)(DA)(DG) (DT)(DC)(DG)(DT)(DT)(DG)(DC)(DT)(DG)(DA) (DT) (DT)(DG)(DG)(DC)(DG)(DT) ...String:
(DG)(DC)(DG)(DT)(DG)(DG)(DC)(DA)(DC)(DA) (DA)(DC)(DA)(DA)(DC)(DT)(DG)(DG)(DC)(DG) (DG)(DG)(DC)(DA)(DA)(DA)(DC)(DA)(DG) (DT)(DC)(DG)(DT)(DT)(DG)(DC)(DT)(DG)(DA) (DT) (DT)(DG)(DG)(DC)(DG)(DT)(DT)(DG) (DC)(DC)(DA)(DC)(DC)(DT)(DC)(DC)(DA)(DG) (DT)(DC) (DT)(DG)(DG)(DC)(DC)(DC)(DT) (DG)(DC)(DA)(DC)(DG)(DC)(DG)(DC)(DC)(DG) (DT)(DC)(DG) (DC)(DA)(DA)(DA)(DT)(DT) (DG)(DT)(DC)(DG)(DC)(DG)(DG)(DC)(DG)(DA) (DT)(DT)(DA)(DA) (DA)(DT)(DC)(DT)(DC) (DG)(DC)(DG)(DC)(DC)(DG)(DA)(DT)(DC)(DA) (DA)(DC)(DT)(DG)(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 / Adenosine diphosphate

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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
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

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6s85

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 199751
FSC plot (resolution estimation)

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