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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-30310 | ||||||||||||
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Title | Human RAD51 post-synaptic complexes mutant (V273P, D274G) | ||||||||||||
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![]() | meitotic homologous recombination / DNA repair / ATPase / RECOMBINATION | ||||||||||||
Function / homology | ![]() presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange ...presynaptic intermediate filament cytoskeleton / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / telomere maintenance via recombination / DNA recombinase assembly / regulation of DNA damage checkpoint / mitotic recombination / Impaired BRCA2 binding to PALB2 / DNA strand invasion / DNA strand exchange activity / reciprocal meiotic recombination / 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 / Homologous DNA Pairing and Strand Exchange / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / single-stranded DNA helicase activity / ATP-dependent DNA damage sensor activity / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / nuclear chromosome / replication fork processing / Transcriptional Regulation by E2F6 / DNA unwinding involved in DNA replication / Presynaptic phase of homologous DNA pairing and strand exchange / ATP-dependent activity, acting on DNA / interstrand cross-link repair / DNA polymerase binding / condensed chromosome / meiotic cell cycle / condensed nuclear chromosome / male germ cell nucleus / cellular response to ionizing radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / site of double-strand break / single-stranded DNA binding / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / mitochondrial matrix / DNA repair / centrosome / DNA damage response / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol Similarity search - Function | ||||||||||||
Biological species | ![]() | ||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.43 Å | ||||||||||||
![]() | Chi HY / Ho MC / Tsai MD / Luo SC / Yeh HY | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures. Authors: Shih-Chi Luo / Hsin-Yi Yeh / Wei-Hsuan Lan / Yi-Min Wu / Cheng-Han Yang / Hao-Yen Chang / Guan-Chin Su / Chia-Yi Lee / Wen-Jin Wu / Hung-Wen Li / Meng-Chiao Ho / Peter Chi / Ming-Daw Tsai / ![]() Abstract: Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and ...Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases. | ||||||||||||
History |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 202.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.8 KB 16.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 13.7 KB | Display | ![]() |
Images | ![]() | 106 KB | ||
Filedesc metadata | ![]() | 6.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 603.7 KB | Display | ![]() |
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Full document | ![]() | 603.2 KB | Display | |
Data in XML | ![]() | 13.4 KB | Display | |
Data in CIF | ![]() | 18.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7c9aMC ![]() 7c98C ![]() 7c99C ![]() 7c9cC ![]() 7cgyC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.84 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : RAD51-dsDNA filament (V273P D274G mutant)
Entire | Name: RAD51-dsDNA filament (V273P D274G mutant) |
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Components |
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-Supramolecule #1: RAD51-dsDNA filament (V273P D274G mutant)
Supramolecule | Name: RAD51-dsDNA filament (V273P D274G mutant) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: DNA repair protein RAD51 homolog 1
Macromolecule | Name: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 36.949074 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ...String: MAMQMQLEAN ADTSVEEESF GPQPISRLEQ CGINANDVKK LEEAGFHTVE AVAYAPKKEL INIKGISEAK ADKILAEAAK LVPMGFTTA TEFHQRRSEI IQITTGSKEL DKLLQGGIET GSITEMFGEF RTGKTQICHT LAVTCQLPID RGGGEGKAMY I DTEGTFRP ERLLAVAERY GLSGSDVLDN VAYARAFNTD HQTQLLYQAS AMMVESRYAL LIVDSATALY RTDYSGRGEL SA RQMHLAR FLRMLLRLAD EFGVAVVITN QVVAQPGGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD UniProtKB: DNA repair protein RAD51 homolog 1 |
-Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
Macromolecule | Name: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 Details: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, ...Details: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 2.692778 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) |
-Macromolecule #3: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
Macromolecule | Name: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 3 Details: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, ...Details: Authors know the sequence of chains D/E. Chain D: TCA GCT GTT GCC CGT. Chain E: ACG GGC AAC AGC TGA. Since the DNA sequence could not be revealed by the helical reconstruction method, authors have used poly-T for chain D, poly-A for chain E respectively for model building. Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 2.773904 KDa |
Sequence | String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) |
-Macromolecule #4: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 3 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ![]() ChemComp-ANP: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Buffer | pH: 7.5 / Component - Concentration: 25.0 mM / Component - Formula: Tris-HCl Details: 25 mM Tris-HCl, pH 7.5, 50 mM KCl and 1 mM dithiothreitol) containing 2 mM AMP-PNP and 5 mM CaCl2 |
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV Details: The grids were blotted for 1 sec at 22 degree C with 100% relative humidity and plunge-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher).. |
Details | protein sample were applied onto a pre-glow-discharged graphene-oxide coated Quantifoil holey carbon grid (1.2/1.3, 200 mesh) using published protocol |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Coma free - Residual tilt: 10.0 mrad |
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 30 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 165000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |