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Yorodumi- PDB-5np7: CryoEM structure of Human Rad51 on single-stranded DNA to 4.2A re... -
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-Basic information
Entry | Database: PDB / ID: 5np7 | ||||||
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Title | CryoEM structure of Human Rad51 on single-stranded DNA to 4.2A resolution. | ||||||
Components | DNA repair protein RAD51 homolog 1 | ||||||
Keywords | RECOMBINATION / recombinase / cryoEM / Human Rad51 / single-stranded DNA | ||||||
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 | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å | ||||||
Authors | Short, J.M. / Venkitaraman, A. | ||||||
Citation | Journal: Nucleic Acids Res / Year: 2016 Title: High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy. Authors: Judith M Short / Yang Liu / Shaoxia Chen / Neelesh Soni / Mallur S Madhusudhan / Mahmud K K Shivji / Ashok R Venkitaraman / Abstract: Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses ...Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5np7.cif.gz | 420.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5np7.ent.gz | 341.2 KB | Display | PDB format |
PDBx/mmJSON format | 5np7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5np7_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 5np7_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 5np7_validation.xml.gz | 76.3 KB | Display | |
Data in CIF | 5np7_validation.cif.gz | 103.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/np/5np7 ftp://data.pdbj.org/pub/pdb/validation_reports/np/5np7 | HTTPS FTP |
-Related structure data
Related structure data | 8183MC 5jzcC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 37009.125 Da / Num. of mol.: 7 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli (E. coli) / References: UniProt: Q06609 #2: Chemical | ChemComp-ANP / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: Helical filament of HRAD51 on single-stranded DNA with AMPPNP Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 259 kDa/nm / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil |
Vitrification | Cryogen name: NITROGEN |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated magnification: 104477 X / Nominal defocus min: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) |
Image scans | Sampling size: 14 µm / Width: 4096 / Height: 4096 / Movie frames/image: 34 |
-Processing
Software | Name: REFMAC / Version: 5.8.0166 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Details: per segment / Type: NONE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 56.2 ° / Axial rise/subunit: 16 Å / Axial symmetry: C1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 70000 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60000 Details: Helical segments were divided in half, not drawn randomly or odd/even as this would result in some segments from the same filament in both half models. Thus, it is strictly not gold-standard. Symmetry type: HELICAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 94.574 / Protocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: Maximum likelihood Details: Backbone fitting is good but many of the sidechains cannot be relied on as the map resolution was insufficient. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 4.2→180 Å / Cor.coef. Fo:Fc: 0.937 / SU B: 46.567 / SU ML: 0.572 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 131.509 Å2
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Refinement step | Cycle: 1 / Total: 16754 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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