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- PDB-8tkq: Cryo-EM structure of human full-length RAD52 -

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Basic information

Entry
Database: PDB / ID: 8tkq
TitleCryo-EM structure of human full-length RAD52
ComponentsDNA repair protein RAD52 homolog
KeywordsRECOMBINATION / DNA repair
Function / homology
Function and homology information


double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination ...double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination / double-strand break repair / single-stranded DNA binding / cellular response to oxidative stress / DNA recombination / protein-containing complex / DNA binding / nucleoplasm / identical protein binding / nucleus
Similarity search - Function
DNA recombination/repair protein Rad52 / DNA repair protein Rad52/59/22 / Rad52 family / DNA repair protein Rad52/59/22 superfamily / Rad52/22 family double-strand break repair protein
Similarity search - Domain/homology
DNA repair protein RAD52 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsSchnicker, N.J. / Razzaghi, M. / Spies, M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA232425-05 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131704-05 United States
CitationJournal: To Be Published
Title: A double-ring of human RAD52 remodels replication forks restricting fork reversal
Authors: Honda, M. / Razzaghi, M.
History
DepositionJul 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair protein RAD52 homolog
B: DNA repair protein RAD52 homolog
C: DNA repair protein RAD52 homolog
D: DNA repair protein RAD52 homolog
E: DNA repair protein RAD52 homolog
F: DNA repair protein RAD52 homolog
G: DNA repair protein RAD52 homolog
H: DNA repair protein RAD52 homolog
I: DNA repair protein RAD52 homolog
J: DNA repair protein RAD52 homolog
K: DNA repair protein RAD52 homolog


Theoretical massNumber of molelcules
Total (without water)532,44411
Polymers532,44411
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Cryo-EM and mass photometry indicated 11-mer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DNA repair protein RAD52 homolog


Mass: 48404.020 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAD52 / Production host: Escherichia coli (E. coli) / References: UniProt: P43351

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human RAD52 undecameric structure / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.532 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 9 / Details: 20 mM Tris pH 9, 200 mM KCl, 1 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2200 mMPotassium chlorideKCl1
31 mMDTT1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was made from frozen protein
Specimen supportDetails: -15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: No tilt data and tilted data (30 deg) was collected at the same time and processed together
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11288

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2261059
Details: Template based picking from no tilt and 30 deg tilt data to deal with preferred orientation
SymmetryPoint symmetry: C11 (11 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 623559 / Symmetry type: POINT
Atomic model buildingB value: 130.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial fitting was done in Chimera followed by refinement with Namdinator and then PHENIX alone
Atomic model buildingPDB-ID: 1KN0

1kn0


Accession code: 1KN0 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00716225
ELECTRON MICROSCOPYf_angle_d0.57421813
ELECTRON MICROSCOPYf_dihedral_angle_d2.589812
ELECTRON MICROSCOPYf_chiral_restr0.0442299
ELECTRON MICROSCOPYf_plane_restr0.0032893

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