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- PDB-5h1c: Human RAD51 post-synaptic complexes -

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Basic information

Entry
Database: PDB / ID: 5h1c
TitleHuman RAD51 post-synaptic complexes
Components
  • DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
  • 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


presynaptic intermediate filament cytoskeleton / 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 / mitotic recombination / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex ...presynaptic intermediate filament cytoskeleton / 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 / mitotic recombination / double-strand break repair involved in meiotic recombination / nuclear ubiquitin ligase complex / DNA strand invasion / replication-born double-strand break repair via sister chromatid exchange / cellular response to hydroxyurea / DNA strand exchange activity / lateral element / telomere maintenance via recombination / regulation of DNA damage checkpoint / Impaired BRCA2 binding to PALB2 / single-stranded DNA helicase 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 / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / nuclear chromosome / replication fork processing / DNA unwinding involved in DNA replication / Transcriptional Regulation by E2F6 / 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 / double-strand break repair via homologous recombination / regulation of protein phosphorylation / HDR through Homologous Recombination (HRR) / PML body / Meiotic recombination / single-stranded DNA binding / site of double-strand break / 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 / cytosol / cytoplasm
Similarity search - Function
DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain ...DNA recombination/repair protein Rad51 / DNA recombination and repair protein, RecA-like / DNA recombination and repair protein Rad51-like, C-terminal / Rad51 / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.5 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 Institute (NIH/NCI)CA168635 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ES007061 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ES015252 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.
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 1.2Oct 18, 2017Group: Author supporting evidence / Data processing / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Mar 23, 2022Group: Author supporting evidence / Database references / Derived calculations
Category: database_2 / pdbx_audit_support / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

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Structure viewerMolecule:
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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')
E: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,08211
Polymers116,4915
Non-polymers1,5926
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15610 Å2
ΔGint-82 kcal/mol
Surface area40280 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
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: DNA chain DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*A)-3')


Mass: 2773.904 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#5: 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

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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 post-synaptic complexCOMPLEX#1-#30MULTIPLE SOURCES
2Human RAD51COMPLEX#11RECOMBINANT
3DNACOMPLEX#21MULTIPLE SOURCES
4DNACOMPLEX#31MULTIPLE SOURCES
Molecular weightValue: 24.18 kDa/nm / Experimental value: NO
Source (recombinant)Organism: human (human) / Plasmid: 1
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 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
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: 528
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: 56.18 ° / Axial rise/subunit: 15.8 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 41146
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30194 / Algorithm: BACK PROJECTION / Num. of class averages: 78 / Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 1SZP

1szp


Pdb chain-ID: E / Pdb chain residue range: 80-395

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