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- PDB-5h1b: Human RAD51 presynaptic complex -

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Basic information

Entry
Database: PDB / ID: 5h1b
TitleHuman RAD51 presynaptic complex
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA repair protein RAD51 homolog 1
KeywordsDNA BINDING PROTEIN/DNA / DNA repair / ATPase / homologous recombination / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


response to glucoside / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / mitotic recombination / telomere maintenance via recombination / positive regulation of DNA ligation / cellular response to cisplatin ...response to glucoside / mitotic recombination-dependent replication fork processing / cellular response to camptothecin / DNA recombinase assembly / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / mitotic recombination / telomere maintenance via recombination / positive regulation of DNA ligation / cellular response to cisplatin / cellular response to hydroxyurea / strand invasion / replication-born double-strand break repair via sister chromatid exchange / lateral element / single-stranded DNA helicase activity / DNA strand exchange activity / reciprocal meiotic recombination / replication fork processing / condensed chromosome / DNA unwinding involved in DNA replication / regulation of double-strand break repair via homologous recombination / response to ionizing radiation / negative regulation of G0 to G1 transition / ATPase, acting on DNA / response to X-ray / DNA polymerase binding / condensed nuclear chromosome / nuclear chromosome / meiotic cell cycle / microtubule organizing center / regulation of protein phosphorylation / double-strand break repair via homologous recombination / cellular response to gamma radiation / cellular response to ionizing radiation / response to toxic substance / PML body / interstrand cross-link repair / site of double-strand break / single-stranded DNA binding / chromatin / chromosome, telomeric region => GO:0000781 / double-stranded DNA binding / protein C-terminus binding / DNA recombination / mitochondrial matrix / chromatin => GO:0000785 / DNA repair / cellular response to DNA damage stimulus / response to drug / chromatin binding / perinuclear region of cytoplasm / enzyme binding / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / identical protein binding / nucleus / cytoplasm
Helix-hairpin-helix domain / DNA recombination and repair protein RecA, monomer-monomer interface / AAA+ ATPase domain / DNA recombination/repair protein Rad51 / DNA recombination and repair protein Rad51-like, C-terminal / DNA recombination and repair protein, RecA-like / DNA repair Rad51/transcription factor NusA, alpha-helical / DNA recombination and repair protein RecA-like, ATP-binding domain / Rad51/DMC1/RadA / Rad51 / P-loop containing nucleoside triphosphate hydrolase
DNA repair protein RAD51 homolog 1
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsXu, J. / Zhao, L. / Xu, Y. / Zhao, W. / Sung, P. / Wang, H.W.
Funding support China, United States, 6items
OrganizationGrant numberCountry
National Science Foundation of China31270765 China
The Key Research and Development Program of MOST2016YFA0501101 China
The Beijing Municipal Science & Technology CommissionZ161100000116034 China
National Institutes of Health/National Cancer InstituteCA168635 United States
National Institutes of Health/National Institute of Environmental Health SciencesES007061 United States
National Institutes of Health/National Institute of Environmental Health SciencesES015252 United States
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structures of human RAD51 recombinase filaments during catalysis of DNA-strand exchange.
Authors: Jingfei Xu / Lingyun Zhao / Yuanyuan Xu / Weixing Zhao / Patrick Sung / Hong-Wei Wang /
Abstract: The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) ...The central step in eukaryotic homologous recombination (HR) is ATP-dependent DNA-strand exchange mediated by the Rad51 recombinase. In this process, Rad51 assembles on single-stranded DNA (ssDNA) and generates a helical filament that is able to search for and invade homologous double-stranded DNA (dsDNA), thus leading to strand separation and formation of new base pairs between the initiating ssDNA and the complementary strand within the duplex. Here, we used cryo-EM to solve the structures of human RAD51 in complex with DNA molecules, in presynaptic and postsynaptic states, at near-atomic resolution. Our structures reveal both conserved and distinct structural features of the human RAD51-DNA complexes compared with their prokaryotic counterpart. Notably, we also captured the structure of an arrested synaptic complex. Our results provide new insight into the molecular mechanisms of the DNA homology search and strand-exchange processes.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 8, 2016Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 21, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2017Group: Database references
Revision 1.2Sep 20, 2017Group: Database references / Category: struct_ref_seq_dif / Item: _struct_ref_seq_dif.details
Revision 1.3Oct 18, 2017Group: Author supporting evidence / Data processing / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization

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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 (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,30910
Polymers113,7174
Non-polymers1,5926
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area13850 Å2
ΔGint-95 kcal/mol
Surface area37780 Å2

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Components

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


Mass: 37008.074 Da / Num. of mol.: 3 / Mutation: K313Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD51, RAD51A, RECA
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q06609
#2: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 2692.778 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Human RAD51 and ssDNA formed presynaptic complexCOMPLEX#1-#20MULTIPLE SOURCES
2Human RAD51COMPLEX#11RECOMBINANT
3ssDNADNACOMPLEX#21MULTIPLE SOURCES
Molecular weightValue: 23.56 kDa/nm / Experimental value: NO
Source (natural)Organism: human (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG / Plasmid: pRh51.2
Buffer solutionpH: 7.5
Details: 25mM Tris-HCl, pH 7.5, 50mM KCl, 1mM dithiothreitol, 1mM AMP-PNP and 2mM MgCl2
SpecimenConc.: 0.075 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 22500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 40404
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 32 / Used frames/image: 3-14

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Processing

EM software
IDNameVersionCategoryDetails
2UCSFImage4image acquisition
4CTFFIND3CTF correction
7Chimera1.9model fitting
9SPIDER20.02initial Euler assignment
10RELION1.2initial Euler assignmentwith helix option
11SPIDER20.02final Euler assignment
12RELION1.2final Euler assignmentwith helix option
13IMAGIC-4D110817classification
14SPIDER20.023D reconstruction
15RELION1.23D reconstructionwith helix option
16Coot0.7.2model refinement
17PHENIX1.9model refinement
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: 56.77 ° / Axial rise/subunit: 15.88 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 540
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33838 / Algorithm: BACK PROJECTION / Num. of class averages: 82 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 1SZP
Pdb chain-ID: E / Pdb chain residue range: 80-395

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