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- 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
TitleCryoEM structure of Human Rad51 on single-stranded DNA to 4.2A resolution.
ComponentsDNA repair protein RAD51 homolog 1
KeywordsRECOMBINATION / recombinase / cryoEM / Human Rad51 / single-stranded DNA
Function/homologyresponse to glucoside / DNA recombination/repair protein Rad51 / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / telomere maintenance via recombination / replication-born double-strand break repair via sister chromatid exchange ...response to glucoside / DNA recombination/repair protein Rad51 / mitotic recombination-dependent replication fork processing / chromosome organization involved in meiotic cell cycle / cellular response to camptothecin / DNA recombinase assembly / telomere maintenance via telomere lengthening / positive regulation of DNA ligation / telomere maintenance via recombination / replication-born double-strand break repair via sister chromatid exchange / mitotic recombination / cellular response to hydroxyurea / single-stranded DNA-dependent ATPase activity / strand invasion / cellular response to cisplatin / DNA recombination and repair protein, RecA-like / lateral element / Rad51/DMC1/RadA / DNA recombination and repair protein Rad51-like, C-terminal / reciprocal meiotic recombination / recombinase activity / strand displacement / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Homologous DNA Pairing and Strand Exchange / Rad51 / DNA unwinding involved in DNA replication / Helix-hairpin-helix domain / Resolution of D-loop Structures through Holliday Junction Intermediates / replication fork processing / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA repair Rad51/transcription factor NusA, alpha-helical / RecA family profile 1. / DNA recombination and repair protein RecA-like, ATP-binding domain / HDR through Single Strand Annealing (SSA) / regulation of double-strand break repair via homologous recombination / DNA synthesis involved in DNA repair / Presynaptic phase of homologous DNA pairing and strand exchange / four-way junction DNA binding / Transcriptional Regulation by E2F6 / negative regulation of G0 to G1 transition / DNA polymerase binding / response to X-ray / site of double-strand break / condensed chromosome / regulation of protein phosphorylation / interstrand cross-link repair / nuclear chromosome / condensed nuclear chromosome / meiotic cell cycle / HDR through Homologous Recombination (HRR) / double-strand break repair via homologous recombination / microtubule organizing center / cellular response to gamma radiation / single-stranded DNA binding / cellular response to ionizing radiation / chromatin / Meiotic recombination / PML body / double-stranded DNA binding / nuclear chromosome, telomeric region / protein homooligomerization / AAA+ ATPase domain / DNA recombination / protein C-terminus binding / nuclear chromatin / mitochondrial matrix / cellular response to DNA damage stimulus / DNA repair / chromatin binding / nucleolus / perinuclear region of cytoplasm / mitochondrion / go:0043234: / P-loop containing nucleoside triphosphate hydrolase / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm / DNA repair protein RAD51 homolog 1
Function and homology information
Specimen sourceHomo sapiens / human /
MethodElectron microscopy (4.2 Å resolution / Helical array / Helical) / Transmission electron microscopy
AuthorsShort, J.M. / Venkitaraman, A.
CitationJournal: 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.
Copyright: The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 13, 2017 / Release: May 3, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 3, 2017Structure modelrepositoryInitial release
1.1Aug 30, 2017Structure modelData collection / Refinement descriptionem_3d_fitting / em_software_em_3d_fitting.target_criteria / _em_software.name
1.2Jan 31, 2018Structure modelDatabase referencespdbx_related_exp_data_set

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Structure visualization

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Assembly

Deposited unit
A: DNA repair protein RAD51 homolog 1
B: DNA repair protein RAD51 homolog 1
C: DNA repair protein RAD51 homolog 1
D: DNA repair protein RAD51 homolog 1
E: DNA repair protein RAD51 homolog 1
F: DNA repair protein RAD51 homolog 1
G: DNA repair protein RAD51 homolog 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)262,60714
Polyers259,0647
Non-polymers3,5437
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)28990
ΔGint (kcal/M)-135
Surface area (Å2)88880
MethodPISA

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Components

#1: Protein/peptide
DNA repair protein RAD51 homolog 1 / hRAD51 / RAD51 homolog A


Mass: 37009.125 Da / Num. of mol.: 7 / Source: (gene. exp.) Homo sapiens / human / / Gene: RAD51, RAD51A, RECA / Production host: Escherichia coli / References: UniProt:Q06609
#2: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 7 / Formula: C10H17N6O12P3 / Comment: AMP-PNP (energy-carrying molecule analogue) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / Reconstruction method: HELICAL

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Sample preparation

ComponentName: Helical filament of HRAD51 on single-stranded DNA with AMPPNP
Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 259 deg. / Units: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 / Grid type: Quantifoil
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 / Calibrated magnification: 104477 / Nominal defocus min: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)
Image scansSampling size: 14 microns / Dimension width: 4096 / Dimension height: 4096 / Movie frames/image: 34

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Processing

SoftwareName: REFMAC / Version: 5.8.0166 / Classification: refinement
EM software
IDNameVersionCategory
1XimdispPARTICLE SELECTION
2EPUIMAGE ACQUISITION
4GctfCTF CORRECTION
7CootMODEL FITTING
11RELION1.4CLASSIFICATION
12RELION1.4RECONSTRUCTION
19REFMACMODEL REFINEMENT
CTF correctionDetails: per segment / Type: NONE
Helical symmertyAngular rotation/subunit: 56.2 deg. / Axial rise/subunit: 16 Å / Axial symmetry: C1
Particle selectionNumber of particles selected: 70000
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number 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 buildingDetails: Backbone fitting is good but many of the sidechains cannot be relied on as the map resolution was insufficient.
Overall b value: 94.574 / Ref protocol: FLEXIBLE FIT / Ref space: RECIPROCAL / Target criteria: Maximum likelihood
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection obsPercent reflection obsOverall SU BOverall SU MLStereochemistry target valuesSolvent model details
1131.509-1.000.210.17-0.54-0.041.540.9370.283150.283154.20180.0063875100.0046.5670.572MAXIMUM LIKELIHOOD WITH PHASESPARAMETERS FOR MASK CACLULATION
ELECTRON MICROSCOPY
Number of atoms included #1Total: 16754
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0190.01917035
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.6471.96623140
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.7245.0002163
ELECTRON MICROSCOPYr_dihedral_angle_2_deg37.02423.434693
ELECTRON MICROSCOPYr_dihedral_angle_3_deg23.00615.0002723
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.90915.000133
ELECTRON MICROSCOPYr_chiral_restr0.2100.2002642
ELECTRON MICROSCOPYr_gen_planes_refined0.0110.02112631
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it11.50012.5798694
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it19.49218.84010843
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it15.79813.9968341
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined44.96760052
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded

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