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- PDB-9bi4: cryo EM structure of dsDNA bound Mre11-Rad50 complex -

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Basic information

Entry
Database: PDB / ID: 9bi4
Titlecryo EM structure of dsDNA bound Mre11-Rad50 complex
Components
  • DNA repair protein RAD50
  • Double-strand break repair protein MRE11
  • one strand of dsDNA
  • second strand of dsDNA
KeywordsDNA BINDING PROTEIN / MRN / Mre11 / Rad50 / Nbs1 / DNA binding / DNA damage / DNA repair
Function / homology
Function and homology information


DNA double-strand break processing involved in repair via synthesis-dependent strand annealing / nucleoside monophosphate phosphorylation / Cytosolic sensors of pathogen-associated DNA / meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / : / mitochondrial double-strand break repair via homologous recombination / DNA Damage/Telomere Stress Induced Senescence / Sensing of DNA Double Strand Breaks / Mre11 complex / chromatin extrusion motor activity ...DNA double-strand break processing involved in repair via synthesis-dependent strand annealing / nucleoside monophosphate phosphorylation / Cytosolic sensors of pathogen-associated DNA / meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / : / mitochondrial double-strand break repair via homologous recombination / DNA Damage/Telomere Stress Induced Senescence / Sensing of DNA Double Strand Breaks / Mre11 complex / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / meiotic DNA double-strand break formation / ascospore formation / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / R-loop processing / Hydrolases; Acting on acid anhydrides / chromosome organization involved in meiotic cell cycle / single-stranded DNA endodeoxyribonuclease activity / homologous chromosome pairing at meiosis / AMP kinase activity / double-stranded telomeric DNA binding / maintenance of DNA trinucleotide repeats / G-quadruplex DNA binding / 3'-5'-DNA exonuclease activity / DNA double-strand break processing / DNA clamp loader activity / single-stranded telomeric DNA binding / telomere maintenance via recombination / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / double-strand break repair via break-induced replication / reciprocal meiotic recombination / mitotic intra-S DNA damage checkpoint signaling / telomeric DNA binding / mitotic G2 DNA damage checkpoint signaling / telomere maintenance via telomerase / 3'-5' exonuclease activity / telomere maintenance / condensed nuclear chromosome / DNA endonuclease activity / meiotic cell cycle / double-strand break repair via homologous recombination / base-excision repair / double-strand break repair via nonhomologous end joining / double-strand break repair / manganese ion binding / site of double-strand break / double-stranded DNA binding / endonuclease activity / molecular adaptor activity / Hydrolases; Acting on ester bonds / chromosome, telomeric region / DNA repair / regulation of transcription by RNA polymerase II / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / metal ion binding / nucleus
Similarity search - Function
DNA repair protein Rad50, eukaryotes / DNA double-strand break repair protein Mre11 / Mre11, DNA-binding / Mre11, capping domain / Mre11 DNA-binding presumed domain / Mre11 DNA-binding presumed domain / Rad50 zinc hook motif / RAD50, zinc hook / Rad50 zinc-hook domain profile. / Mre11 nuclease, N-terminal metallophosphatase domain ...DNA repair protein Rad50, eukaryotes / DNA double-strand break repair protein Mre11 / Mre11, DNA-binding / Mre11, capping domain / Mre11 DNA-binding presumed domain / Mre11 DNA-binding presumed domain / Rad50 zinc hook motif / RAD50, zinc hook / Rad50 zinc-hook domain profile. / 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
ADENOSINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / DNA repair protein RAD50 / Double-strand break repair protein MRE11
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYu, Y. / Patel, D.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136278 United States
CitationJournal: Res Sq / Year: 2024
Title: Structure guided functional analysis of the S. cerevisiae Mre11 complex.
Authors: John Petrini / Marcel Hohl / You Yu / Vitaly Kuryavyi / Dinshaw Patel
Abstract: The Mre11 complex comprises Mre11, Rad50 and Nbs1 (Xrs2 in ). The core components, Mre11 and Rad50 are highly conserved, with readily identifiable orthologs in all clades of life, whereas Nbs1/Xrs2 ...The Mre11 complex comprises Mre11, Rad50 and Nbs1 (Xrs2 in ). The core components, Mre11 and Rad50 are highly conserved, with readily identifiable orthologs in all clades of life, whereas Nbs1/Xrs2 are present only in eukaryotes. In eukaryotes, the complex is integral to the DNA damage response, acting in DNA double strand break (DSB) detection and repair, and the activation of DNA damage signaling. We present here a 3.2 Å cryo-EM structure of the Mre11-Rad50 complex with bound dsDNA. The structure provided a foundation for detailed mutational analyses regarding homo and heterotypic protein interfaces, as well as DNA binding properties of Rad50. We define several conserved residues in Rad50 and Mre11 that are critical to complex assembly as well as for DNA binding. In addition, the data reveal that the Rad50 coiled coil domain influences ATP hydrolysis over long distances.
History
DepositionApr 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA repair protein RAD50
D: DNA repair protein RAD50
B: Double-strand break repair protein MRE11
E: one strand of dsDNA
A: Double-strand break repair protein MRE11
F: second strand of dsDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)517,24714
Polymers515,9646
Non-polymers1,2838
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules CDBA

#1: Protein DNA repair protein RAD50 / 153 kDa protein


Mass: 152797.688 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAD50, YNL250W, N0872 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P12753, Hydrolases; Acting on acid anhydrides
#2: Protein Double-strand break repair protein MRE11


Mass: 79607.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MRE11, YMR224C, YM9959.06C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32829

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DNA chain , 2 types, 2 molecules EF

#3: DNA chain one strand of dsDNA


Mass: 25951.223 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain second strand of dsDNA


Mass: 25203.014 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 8 molecules

#5: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: Mg
#7: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: dsDNA bound yeast Mre11-Rad50 complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.46 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Details: 50 mM Hepes, pH 7.5, 200 mM NaCl, 2 mM DTT, 2% Glycerol, 0.025% (w/v) CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate)
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 51.22 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 242558 / Symmetry type: POINT

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