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Yorodumi- EMDB-16170: CryoEM structure of the presynaptic RAD51 nucleoprotein filament ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16170 | |||||||||
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Title | CryoEM structure of the presynaptic RAD51 nucleoprotein filament in the presence of ATP and Ca2+ | |||||||||
Map data | postprocessed map | |||||||||
Sample |
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Keywords | DNA repair / Homologous Recombination / DNA-strand exchange / ATPase / DNA BINDING PROTEIN | |||||||||
Function / homology | Function and homology information 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 / nuclear ubiquitin ligase complex / double-strand break repair involved in meiotic recombination / 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 / nuclear ubiquitin ligase complex / double-strand break repair involved in meiotic recombination / cellular response to hydroxyurea / replication-born double-strand break repair via sister chromatid exchange / lateral element / DNA recombinase assembly / telomere maintenance via recombination / regulation of DNA damage checkpoint / mitotic recombination / DNA strand invasion / Impaired BRCA2 binding to PALB2 / DNA strand exchange activity / reciprocal meiotic recombination / single-stranded DNA helicase activity / 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 / 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 / condensed chromosome / DNA unwinding involved in DNA replication / replication fork processing / Transcriptional Regulation by E2F6 / Presynaptic phase of homologous DNA pairing and strand exchange / condensed nuclear chromosome / ATP-dependent activity, acting on DNA / meiotic cell cycle / male germ cell nucleus / interstrand cross-link repair / DNA polymerase binding / Meiotic recombination / cellular response to ionizing radiation / regulation of protein phosphorylation / double-strand break repair via homologous recombination / HDR through Homologous Recombination (HRR) / PML body / double-stranded DNA binding / site of double-strand break / single-stranded DNA binding / chromosome, telomeric region / DNA recombination / mitochondrial matrix / DNA repair / chromatin binding / centrosome / DNA damage response / 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 | Homo sapiens (human) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Appleby R / Bollschweiler D / Pellegrini L | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: iScience / Year: 2023 Title: A metal ion-dependent mechanism of RAD51 nucleoprotein filament disassembly. Authors: Robert Appleby / Daniel Bollschweiler / Dimitri Y Chirgadze / Luay Joudeh / Luca Pellegrini / Abstract: The RAD51 ATPase polymerizes on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of homologous recombination. ATP binding maintains the NPF ...The RAD51 ATPase polymerizes on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the reaction of homologous recombination. ATP binding maintains the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, contributing to NPF disassembly. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16170.map.gz | 28.2 MB | EMDB map data format | |
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Header (meta data) | emd-16170-v30.xml emd-16170.xml | 17.8 KB 17.8 KB | Display Display | EMDB header |
Images | emd_16170.png | 68.3 KB | ||
Filedesc metadata | emd-16170.cif.gz | 6.2 KB | ||
Others | emd_16170_half_map_1.map.gz emd_16170_half_map_2.map.gz | 22.9 MB 22.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16170 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16170 | HTTPS FTP |
-Validation report
Summary document | emd_16170_validation.pdf.gz | 780.2 KB | Display | EMDB validaton report |
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Full document | emd_16170_full_validation.pdf.gz | 779.8 KB | Display | |
Data in XML | emd_16170_validation.xml.gz | 10.6 KB | Display | |
Data in CIF | emd_16170_validation.cif.gz | 12.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16170 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16170 | HTTPS FTP |
-Related structure data
Related structure data | 8bq2MC 8br2C 8bscC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_16170.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | postprocessed map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.43 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: half map 2
File | emd_16170_half_map_1.map | ||||||||||||
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Annotation | half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 1
File | emd_16170_half_map_2.map | ||||||||||||
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Annotation | half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Nucleoprotein filament of human RAD51 bound to single-stranded DN...
Entire | Name: Nucleoprotein filament of human RAD51 bound to single-stranded DNA 60mer in the presence of ATP and Ca2+ |
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Components |
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-Supramolecule #1: Nucleoprotein filament of human RAD51 bound to single-stranded DN...
Supramolecule | Name: Nucleoprotein filament of human RAD51 bound to single-stranded DNA 60mer in the presence of ATP and Ca2+ type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2 Details: sample was prepared by mixing RAD51, DNA, ATP in Ca2+ buffer |
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Source (natural) | Organism: Homo sapiens (human) |
-Macromolecule #1: DNA repair protein RAD51 homolog 1
Macromolecule | Name: DNA repair protein RAD51 homolog 1 / type: protein_or_peptide / ID: 1 / Details: RAD51 protein with bound ATP and Ca2+ / Number of copies: 9 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 37.009125 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
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 QVVAQVDGAA MFAADPKKPI GGNIIAHAST TRLYLRKGRG ETRICKIYDS PCL PEAEAM FAINADGVGD AKD UniProtKB: DNA repair protein RAD51 homolog 1 |
-Macromolecule #2: DNA (30-MER)
Macromolecule | Name: DNA (30-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 9.671227 KDa |
Sequence | String: (DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG) (DG)(DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG) (DA)(DG)(DG)(DA)(DG)(DG)(DA)(DG)(DG) (DA) |
-Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 9 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Macromolecule #4: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 18 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING |
Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1339 / Average exposure time: 2.0 sec. / Average electron dose: 66.0 e/Å2 Details: Images were collected in movie-mode at 38 frames/movie. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Helical parameters - Δz: 16.6 Å Applied symmetry - Helical parameters - Δ&Phi: 56.7 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 723142 |
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Segment selection | Number selected: 850217 / Software - Name: RELION (ver. 3.0) / Details: Segments were picked using Autopicking in RELION. |
Startup model | Type of model: OTHER Details: An initial 3D model was generated from 5000 selected particles in RELION and low-pass filtered at 40A for 3D classification. |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: RELION (ver. 3.0) |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: OTHER |
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Output model | PDB-8bq2: |