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- PDB-8wt9: Cryo-EM structure of the IS621 recombinase in complex with bridge... -

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Basic information

Entry
Database: PDB / ID: 8wt9
TitleCryo-EM structure of the IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the post-strand exchange state (Holliday junction resolution)
Components
  • IS621 transposase
  • bridge RNA
  • donor DNA
  • donor DNA-target DNA
  • target DNA
  • target DNA-donor DNA
KeywordsRECOMBINATION/RNA/DNA / Holliday junction / RNA dependent recombinase / 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: Masahiro Hiraizumi / Nicholas T Perry / Matthew G Durrant / Teppei Soma / Naoto Nagahata / Sae Okazaki / Januka S Athukoralage / Yukari Isayama / James J Pai / April Pawluk / Silvana ...Authors: Masahiro Hiraizumi / Nicholas T Perry / Matthew G Durrant / Teppei Soma / Naoto Nagahata / Sae Okazaki / Januka S Athukoralage / Yukari Isayama / James J Pai / April Pawluk / Silvana Konermann / Keitaro Yamashita / Patrick D Hsu / Hiroshi Nishimasu /
Abstract: Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes. We recently discovered that IS110 family elements encode a recombinase and a non- ...Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC-Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5'-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.
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
Revision 1.2Jul 17, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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-donor DNA
H: target DNA
I: donor DNA-target DNA
J: donor DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)312,78612
Polymers312,73810
Non-polymers492
Water362
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-donor DNA


Mass: 15003.622 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: DNA chain target DNA


Mass: 11703.487 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The linkage between 17H and 18H has been cleaved by IS621.
Source: (synth.) Escherichia coli (E. coli)
#5: DNA chain donor DNA-target DNA


Mass: 10163.561 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain donor DNA


Mass: 13587.737 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The linkage between 28J and 29J has been cleaved by IS621.
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, 4 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.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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 post-strand exchange state (Holliday junction resolution)
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: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 62 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: 752628 / Symmetry type: POINT

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