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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 / homology
Function and homology information


presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to cisplatin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / DNA strand invasion / mitotic recombination ...presynaptic intermediate filament cytoskeleton / response to glucoside / mitotic recombination-dependent replication fork processing / DNA recombinase assembly / cellular response to cisplatin / chromosome organization involved in meiotic cell cycle / telomere maintenance via telomere lengthening / double-strand break repair involved in meiotic recombination / DNA strand invasion / mitotic recombination / cellular response to camptothecin / lateral element / DNA strand exchange activity / Impaired BRCA2 binding to PALB2 / regulation of DNA damage checkpoint / telomere maintenance via recombination / reciprocal meiotic recombination / single-stranded DNA helicase activity / ATP-dependent DNA damage sensor activity / regulation of double-strand break repair via homologous recombination / HDR through Single Strand Annealing (SSA) / nuclear chromosome / Homologous DNA Pairing and Strand Exchange / 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 / Resolution of D-loop Structures through Synthesis-Dependent Strand Annealing (SDSA) / Resolution of D-loop Structures through Holliday Junction Intermediates / Transcriptional Regulation by E2F6 / Impaired BRCA2 binding to RAD51 / replication fork processing / Presynaptic phase of homologous DNA pairing and strand exchange / response to X-ray / ATP-dependent activity, acting on DNA / interstrand cross-link repair / condensed chromosome / DNA polymerase binding / condensed nuclear chromosome / cellular response to ionizing radiation / meiotic cell cycle / cellular response to gamma radiation / protein-DNA complex / PML body / double-strand break repair via homologous recombination / Meiotic recombination / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / response to toxic substance / HDR through Homologous Recombination (HRR) / single-stranded DNA binding / site of double-strand break / double-stranded DNA binding / DNA recombination / chromosome, telomeric region / response to xenobiotic stimulus / mitochondrial matrix / hydrolase activity / DNA repair / chromatin binding / centrosome / DNA damage response / nucleolus / chromatin / perinuclear region of cytoplasm / enzyme binding / 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 ...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 / SAM-like Helix-hairpin-helix tandem / 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 repair protein RAD51 homolog 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
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.
History
DepositionApr 13, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software / Item: _em_3d_fitting.target_criteria / _em_software.name
Revision 1.2Jan 31, 2018Group: Database references / Category: pdbx_related_exp_data_set
Revision 1.3Oct 17, 2018Group: Data collection / Other / Refinement description / Category: cell / refine
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
Polymers259,0647
Non-polymers3,5437
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area28990 Å2
ΔGint-135 kcal/mol
Surface area88880 Å2
MethodPISA

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Components

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


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 / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 7 / 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: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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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 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: NITROGEN

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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 FIELD / Nominal magnification: 59000 X / Calibrated magnification: 104477 X / 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 µm / Width: 4096 / 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
12RELION1.43D reconstruction
19REFMACmodel refinement
CTF correctionDetails: per segment / Type: NONE
Helical symmertyAngular rotation/subunit: 56.2 ° / Axial rise/subunit: 16 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 70000
3D reconstructionResolution: 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 buildingB 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.
RefinementResolution: 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
RfactorNum. reflection% reflection
Rwork0.28315 --
obs0.28315 63875 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 131.509 Å2
Baniso -1Baniso -2Baniso -3
1--1 Å20.21 Å20.17 Å2
2---0.54 Å2-0.04 Å2
3---1.54 Å2
Refinement stepCycle: 1 / Total: 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_deg6.72452163
ELECTRON MICROSCOPYr_dihedral_angle_2_deg37.02423.434693
ELECTRON MICROSCOPYr_dihedral_angle_3_deg23.006152723
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.90915133
ELECTRON MICROSCOPYr_chiral_restr0.210.22642
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_it11.512.5798694
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it19.49218.8410843
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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