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- PDB-8h1p: Cryo-EM structure of the human RAD52 protein -

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Basic information

Entry
Database: PDB / ID: 8h1p
TitleCryo-EM structure of the human RAD52 protein
ComponentsDNA repair protein RAD52 homolog
KeywordsRECOMBINATION / double-strand break repair / single strand annealing protein / DNA binding protein / self-oligomerization
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: 3.48 Å
AuthorsKinoshita, C. / Takizawa, Y. / Saotome, M. / Ogino, S. / Kurumizaka, H. / Kagawa, W.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP18H05534 Japan
Japan Society for the Promotion of Science (JSPS)19K12328 Japan
Japan Society for the Promotion of Science (JSPS)20H00449 Japan
Japan Society for the Promotion of Science (JSPS)22H03743 Japan
Japan Society for the Promotion of Science (JSPS)22K06098 Japan
Japan Science and TechnologyJPMJER1901 Japan
Japan Agency for Medical Research and Development (AMED)JP22am121009 Japan
CitationJournal: FEBS Open Bio / Year: 2023
Title: The cryo-EM structure of full-length RAD52 protein contains an undecameric ring.
Authors: Chiaki Kinoshita / Yoshimasa Takizawa / Mika Saotome / Shun Ogino / Hitoshi Kurumizaka / Wataru Kagawa /
Abstract: The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and ...The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N-terminal half. However, only low-resolution structures have been reported for the full-length protein, and thus the structural role of the C-terminal half in self-oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo-electron microscopy (cryo-EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N-terminal half. The cryo-EM map for the C-terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo-EM structure provides important insights into the mechanistic roles played by the N-terminal and C-terminal halves of RAD52 during DNA double-strand break repair.
History
DepositionOct 3, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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)511,66411
Polymers511,66411
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DNA repair protein RAD52 homolog /


Mass: 46514.934 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: RAD52 / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
20.1 mM2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acidEDTAEthylenediaminetetraacetic acid1
32 mM2-mercaptoethanol2-mercaptoethanol1
40.4 Mpotassium chlorideKCl1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil 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: 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: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1.14CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39100 / Algorithm: BACK PROJECTION / Symmetry type: POINT

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