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- PDB-9q9m: Cryo-EM structure of human Mre11-Rad50-Nbs1 (MRN) complex bound t... -

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Basic information

Entry
Database: PDB / ID: 9q9m
TitleCryo-EM structure of human Mre11-Rad50-Nbs1 (MRN) complex bound to DNA and telomeric factor TRF2
Components
  • (DNA (64-MER)) x 2
  • DNA repair protein RAD50
  • Double-strand break repair protein MRE11
  • Nibrin
  • Telomeric repeat-binding factor 2
KeywordsHYDROLASE / Mre11-Rad50-Nbs1-TRF2 complex / double-strand DNA break repair protein / nuclease
Function / homology
Function and homology information


telomere maintenance via telomere trimming / chromosomal region / axonal transport of messenger ribonucleoprotein complex / negative regulation of telomere single strand break repair / telomeric 3' overhang formation / negative regulation of telomere maintenance via recombination / telomeric loop formation / mitochondrial double-strand break repair via homologous recombination / Mre11 complex / negative regulation of telomere maintenance via semi-conservative replication ...telomere maintenance via telomere trimming / chromosomal region / axonal transport of messenger ribonucleoprotein complex / negative regulation of telomere single strand break repair / telomeric 3' overhang formation / negative regulation of telomere maintenance via recombination / telomeric loop formation / mitochondrial double-strand break repair via homologous recombination / Mre11 complex / negative regulation of telomere maintenance via semi-conservative replication / negative regulation of telomeric D-loop disassembly / negative regulation of double-strand break repair via nonhomologous end joining / blastocyst growth / negative regulation of telomere capping / BRCA1-C complex / Sensing of DNA Double Strand Breaks / meiotic DNA double-strand break formation / protection from non-homologous end joining at telomere / regulation of mitotic recombination / RNA-templated DNA biosynthetic process / R-loop processing / Hydrolases; Acting on acid anhydrides / negative regulation of telomere maintenance / t-circle formation / negative regulation of t-circle formation / telomeric D-loop disassembly / single-stranded DNA endodeoxyribonuclease activity / chromosome organization involved in meiotic cell cycle / shelterin complex / phosphorylation-dependent protein binding / Telomere C-strand synthesis initiation / homologous chromosome pairing at meiosis / regulation of telomere maintenance via telomerase / double-stranded telomeric DNA binding / DNA strand resection involved in replication fork processing / nuclease activity / homologous recombination / G-quadruplex DNA binding / nuclear inclusion body / 3'-5'-DNA exonuclease activity / DNA double-strand break processing / Telomere C-strand (Lagging Strand) Synthesis / single-stranded telomeric DNA binding / nuclear telomere cap complex / Impaired BRCA2 binding to PALB2 / double-strand break repair via alternative nonhomologous end joining / chromatin-protein adaptor activity / G-rich strand telomeric DNA binding / protein localization to site of double-strand break / telomere capping / telomere maintenance via recombination / isotype switching / Cytosolic sensors of pathogen-associated DNA / mitotic G2/M transition checkpoint / HDR through MMEJ (alt-NHEJ) / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / IRF3-mediated induction of type I IFN / Removal of the Flap Intermediate from the C-strand / reciprocal meiotic recombination / mitotic intra-S DNA damage checkpoint signaling / regulation of telomere maintenance / regulation of DNA-templated DNA replication initiation / negative regulation of telomere maintenance via telomere lengthening / sister chromatid cohesion / Homologous DNA Pairing and Strand Exchange / Defective homologous recombination repair (HRR) due to BRCA1 loss of function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA1 binding function / Defective HDR through Homologous Recombination Repair (HRR) due to PALB2 loss of BRCA2/RAD51/RAD51C binding function / protein localization to chromosome, telomeric region / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / HDR through Single Strand Annealing (SSA) / neuromuscular process controlling balance / positive regulation of double-strand break repair / telomeric DNA binding / Impaired BRCA2 binding to RAD51 / negative regulation of telomere maintenance via telomerase / mitotic G2 DNA damage checkpoint signaling / positive regulation of telomere maintenance / telomere maintenance in response to DNA damage / Presynaptic phase of homologous DNA pairing and strand exchange / negative regulation of cellular senescence / Telomere Extension By Telomerase / protein K63-linked ubiquitination / neuroblast proliferation / telomere maintenance via telomerase / positive regulation of double-strand break repair via homologous recombination / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / 3'-5' exonuclease activity / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / telomere maintenance / Inhibition of DNA recombination at telomere / intrinsic apoptotic signaling pathway / Meiotic synapsis / DNA damage checkpoint signaling / replication fork
Similarity search - Function
Nibrin, C-terminal / Nibrin / DNA damage repair protein Nbs1 / DNA damage repair protein Nbs1 / Nibrin, second BRCT domain / Nibrin, second BRCT domain superfamily / Second BRCT domain on Nijmegen syndrome breakage protein / DNA repair protein Rad50, eukaryotes / Nibrin-related / Telomeric repeat-binding factor 2, Rap1-binding domain ...Nibrin, C-terminal / Nibrin / DNA damage repair protein Nbs1 / DNA damage repair protein Nbs1 / Nibrin, second BRCT domain / Nibrin, second BRCT domain superfamily / Second BRCT domain on Nijmegen syndrome breakage protein / DNA repair protein Rad50, eukaryotes / Nibrin-related / Telomeric repeat-binding factor 2, Rap1-binding domain / Telomeric repeat-binding factor 2 Rap1-binding motif / Telomeric repeat-binding factor 2 / 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. / Telomeric repeat-binding factor 1/2 / Telomere repeat-binding factor, dimerisation domain superfamily / Telomere repeat-binding factor, dimerisation domain / Telomere repeat binding factor (TRF) / Mre11 nuclease, N-terminal metallophosphatase domain / SbcC/RAD50-like, Walker B motif / Rad50/SbcC-type AAA domain / AAA domain / Myb-type HTH DNA-binding domain profile. / Forkhead associated domain / Forkhead-associated (FHA) domain profile. / FHA domain / Forkhead-associated (FHA) domain / Myb domain / Myb-like DNA-binding domain / SMAD/FHA domain superfamily / BRCA1 C Terminus (BRCT) domain / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / BRCT domain / BRCT domain superfamily / Homeobox-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / BERYLLIUM TRIFLUORIDE ION / : / DNA / DNA (> 10) / Nibrin / Double-strand break repair protein MRE11 / Telomeric repeat-binding factor 2 / DNA repair protein RAD50
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsCui, H.J. / Lammens, K. / Hopfner, K.P. / Fan, Y.L. / Kuybu, F.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for DNA break sensing by human MRE11-RAD50-NBS1 and its regulation by telomeric factor TRF2.
Authors: Yilan Fan / Filiz Kuybu / Hengjun Cui / Katja Lammens / Jia-Xuan Chen / Michael Kugler / Christophe Jung / Karl-Peter Hopfner /
Abstract: The MRE11-RAD50-NBS1 (MRN) complex is a central, multifunctional factor in the detection, signaling and nucleolytic processing of DNA double-strand breaks (DSBs). To clarify how human MRN binds ...The MRE11-RAD50-NBS1 (MRN) complex is a central, multifunctional factor in the detection, signaling and nucleolytic processing of DNA double-strand breaks (DSBs). To clarify how human MRN binds generic and telomeric DNA ends and can separate DNA end sensing from nuclease activities, we determined cryo-electron microscopy (cryo-EM) structures of human MRN bound to DNA and to DNA and the telomere protection factor TRF2. MRN senses DSBs through a tight clamp-like sensing state with closed coiled-coil domains, but auto-inhibited MRE11 nuclease. NBS1 wraps around the MRE11 dimer, with NBS1's ATM recruitment motif sequestered by binding to the regulatory RAD50 S site, necessitating a switch in the NBS1 C helix for ATM activation. At telomeric DNA, TRF2 blocks the second S site via the iDDR motif to prevent nuclease and ATM activation. Our results provide a structural framework for DNA sensing via a gating mechanism and separation of sensing, signaling and processing activities of mammalian MRN.
History
DepositionFeb 26, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
J: Telomeric repeat-binding factor 2
P: DNA (64-MER)
T: DNA (64-MER)
F: Nibrin
A: DNA repair protein RAD50
B: DNA repair protein RAD50
D: Double-strand break repair protein MRE11
E: Double-strand break repair protein MRE11
hetero molecules


Theoretical massNumber of molelcules
Total (without water)663,81218
Polymers662,5578
Non-polymers1,25510
Water1086
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 6 molecules JFABDE

#1: Protein Telomeric repeat-binding factor 2 / TTAGGG repeat-binding factor 2 / Telomeric DNA-binding protein


Mass: 61694.660 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The N-terminal GPASG sequence is the leftover from PreScission cleavage site, while the C-terminal SLEHHHHHHHHHH sequence resulted from His10 purification tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: TERF2, TRBF2, TRF2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15554
#4: Protein Nibrin / Cell cycle regulatory protein p95 / Nijmegen breakage syndrome protein 1


Mass: 85073.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NBN, NBS, NBS1, P95 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O60934
#5: Protein DNA repair protein RAD50


Mass: 154150.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD50 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92878
#6: Protein Double-strand break repair protein MRE11 / Meiotic recombination 11 homolog 1 / MRE11 homolog 1 / Meiotic recombination 11 homolog A / MRE11 homolog A


Mass: 84032.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Following residue R708 is a short GS linker, a PreScission cleavage site, and two FLAG tags.
Source: (gene. exp.) Homo sapiens (human) / Gene: MRE11, HNGS1, MRE11A / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P49959, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules PT

#2: DNA chain DNA (64-MER)


Mass: 19423.369 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain DNA (64-MER)


Mass: 20000.281 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 5 types, 16 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#9: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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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: MRN-TRF2-DNA complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.652 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Details: 25mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM ATP, 1mM BeF3, 5 mM MgCl2, 1 mM MnCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA, 7 s / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.2particle selection
2EPU3.9.1.8206image acquisition
4cryoSPARC4.6.2CTF correction
7UCSF ChimeraX1.9model fitting
9PHENIX1.20.1model refinement
10cryoSPARC4.6.2initial Euler assignment
11cryoSPARC4.6.2final Euler assignment
12cryoSPARC4.6.2classification
13cryoSPARC4.6.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208916 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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