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- PDB-8wt7: Cryo-EM structure of the IS621 recombinase in complex with bridge... -

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Basic information

Entry
Database: PDB / ID: 8wt7
TitleCryo-EM structure of the IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the pre-strand exchange locked state
Components
  • (donor DNA) x 2
  • (target DNA) x 2
  • IS621 transposase
  • bridge RNA
KeywordsRECOMBINATION/RNA/DNA / Holliday junction / RNA dependent recombinase / RECOMBINATION / RECOMBINATION-RNA-DNA complex
Function / homology
Function and homology information


transposase activity / DNA transposition / DNA binding
Similarity search - Function
Transposase, IS111A/IS1328/IS1533, N-terminal / Transposase, IS116/IS110/IS902 / : / Transposase / Transposase IS116/IS110/IS902 family
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / IS621 transposase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHiraizumi, M. / Yamashita, K. / Nishimasu, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR23B6 Japan
Other privateInamori Foundation
CitationJournal: Nature / Year: 2024
Title: Structural mechanism of bridge RNA-guided recombination
Authors: Hiraizumi, M. / Perry, N.T. / Durrant, M.G. / Soma, T. / Nagahata, N. / Okazaki, S. / Athukoralage, J.S. / Isayama, Y. / Pai, J.J. / Pawluk, A. / Konermann, S. / Yamashita, K. / Hsu, P.D. / Nishimasu, H.
History
DepositionOct 18, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: IS621 transposase
B: IS621 transposase
C: IS621 transposase
D: IS621 transposase
E: bridge RNA
F: bridge RNA
G: target DNA
H: target DNA
I: donor DNA
J: donor DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,78612
Polymers312,73810
Non-polymers492
Water724
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 4 types, 4 molecules GHIJ

#3: DNA chain target DNA


Mass: 11659.493 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The linkage between 25G and 26G has been cleaved by IS621.
Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain target DNA


Mass: 11703.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#5: DNA chain donor DNA


Mass: 13507.688 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The linkage between 20I and 21I has been cleaved by IS621.
Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain donor DNA


Mass: 13587.737 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / RNA chain , 2 types, 6 molecules ABCDEF

#1: Protein
IS621 transposase


Mass: 36735.355 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: O3K_03330, O3K_03970, O3K_05990, O3K_07835, O3K_09380, O3K_16710, O3K_17245, O3K_20600, O3K_22425
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0E0Y1P1
#2: RNA chain bridge RNA


Mass: 57668.918 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: synthetic construct (others)

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Non-polymers , 2 types, 6 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: The IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the pre-strand exchange locked state
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 49.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
9Servalcatmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 875289 / Symmetry type: POINT

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