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- EMDB-37829: Cryo-EM structure of the IS621 recombinase in complex with bridge... -

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Basic information

Entry
Database: EMDB / ID: EMD-37829
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 intermediate)
Map data
Sample
  • Complex: The IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the post-strand exchange state (Holliday junction intermediate)
    • Protein or peptide: IS621 transposase
    • RNA: bridge RNA
    • DNA: target DNA-donor DNA
    • DNA: target DNA
    • DNA: donor DNA-target DNA
    • DNA: donor DNA
  • Ligand: MAGNESIUM ION
KeywordsHolliday junction / RNA dependent recombinase / RECOMBINATION-RNA-DNA COMPLEX
Function / homologyTransposase, IS111A/IS1328/IS1533, N-terminal / : / Transposase / Transposase, IS116/IS110/IS902 / Transposase IS116/IS110/IS902 family / transposase activity / DNA transposition / DNA binding / IS621 transposase
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsHiraizumi M / Yamashita K / Nishimasu H
Funding support Japan, 2 items
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, 2023-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37829.png

Downloads & links

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Map

FileDownload / File: emd_37829.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 240 pix.
= 265.6 Å		1.11 Å/pix.
x 240 pix.
= 265.6 Å		1.11 Å/pix.
x 240 pix.
= 265.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.10667 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.34007472 - 0.84418106
Average (Standard dev.)-0.0008846668 (±0.025724959)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 265.6001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37829_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_37829_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37829_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : The IS621 recombinase in complex with bridge RNA, donor DNA, and ...

EntireName: The IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the post-strand exchange state (Holliday junction intermediate)
Components
  • Complex: The IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the post-strand exchange state (Holliday junction intermediate)
    • Protein or peptide: IS621 transposase
    • RNA: bridge RNA
    • DNA: target DNA-donor DNA
    • DNA: target DNA
    • DNA: donor DNA-target DNA
    • DNA: donor DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The IS621 recombinase in complex with bridge RNA, donor DNA, and ...

SupramoleculeName: The IS621 recombinase in complex with bridge RNA, donor DNA, and target DNA in the post-strand exchange state (Holliday junction intermediate)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: IS621 transposase

MacromoleculeName: IS621 transposase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 36.735355 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GPMEHELHYI GIDTAKEKLD VDVLRPDGRH RTKKFANTTK GHDELVSWLK GHKIDHAHIC IEATGTYMEP VAECLYDAGY IVSVINPAL GKAFAQSEGL RNKTDTVDAR MLAEFCRQKR PAAWEAPHPL ERALRALVVR HQALTDMHTQ ELNRTETARE V QRPSIDAH ...String:
GPMEHELHYI GIDTAKEKLD VDVLRPDGRH RTKKFANTTK GHDELVSWLK GHKIDHAHIC IEATGTYMEP VAECLYDAGY IVSVINPAL GKAFAQSEGL RNKTDTVDAR MLAEFCRQKR PAAWEAPHPL ERALRALVVR HQALTDMHTQ ELNRTETARE V QRPSIDAH LLWLEAELKR LEKQIKDLTD DDPDMKHRRK LLESIPGIGE KTSAVLLAYI GLKDRFAHAR QFAAFAGLTP RR YESGSSV RGASRMSKAG HVSLRRALYM PAMVATSKTE WGRAFRDRLA ANGKKGKVIL GAMMRKLAQV AYGVLKSGVP FDA SRHNPV AA

UniProtKB: IS621 transposase

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Macromolecule #2: bridge RNA

MacromoleculeName: bridge RNA / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 57.668918 KDa
SequenceString:
GGGAGUGCAG AGAAAAUCGG CCAGUUUUCU CUGCCUGCAG UCCGCAUGCC GUAUCGGGCC UUGGGUUCUA ACCUGUUCUG UAGAUUUAU GCAGCGGACU GCCUUUCUCC CAAAGUGAUA AACCGGACAG UAUCAUGGAC CGGUUUUCCC GGUAAUCCGU A UUUACAAG GCUGGUUUCA CU

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Macromolecule #3: target DNA-donor DNA

MacromoleculeName: target DNA-donor DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 15.003622 KDa
SequenceString:
(DG)(DC)(DC)(DG)(DG)(DG)(DT)(DA)(DA)(DT) (DA)(DC)(DC)(DA)(DC)(DC)(DA)(DA)(DG)(DC) (DC)(DG)(DC)(DC)(DT)(DT)(DG)(DT)(DA) (DT)(DT)(DA)(DT)(DC)(DC)(DC)(DT)(DC)(DC) (DA) (DG)(DT)(DG)(DC)(DA)(DG)(DA)(DG) (DA)

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Macromolecule #4: target DNA

MacromoleculeName: target DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.703487 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DA)(DT)(DC) (DT)(DG)(DT)(DA)(DG)(DG)(DC)(DC)(DC)(DG) (DA)(DT)(DG)(DG)(DT)(DG)(DG)(DT)(DA) (DT)(DT)(DA)(DC)(DC)(DC)(DG)(DG)(DC)

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Macromolecule #5: donor DNA-target DNA

MacromoleculeName: donor DNA-target DNA / type: dna / ID: 5
Details: The linkage between 20I and 21I has been cleaved by IS621.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.163561 KDa
SequenceString:
(DT)(DG)(DC)(DA)(DG)(DG)(DC)(DC)(DA)(DT) (DA)(DA)(DG)(DT)(DC)(DA)(DA)(DT)(DC)(DT) (DA)(DC)(DA)(DG)(DA)(DT)(DG)(DA)(DG) (DC)(DT)(DC)(DG)

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Macromolecule #6: donor DNA

MacromoleculeName: donor DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 13.587737 KDa
SequenceString:
(DT)(DC)(DT)(DC)(DT)(DG)(DC)(DA)(DC)(DT) (DG)(DG)(DA)(DG)(DG)(DG)(DA)(DT)(DA)(DA) (DT)(DA)(DC)(DA)(DA)(DG)(DA)(DT)(DA) (DC)(DT)(DG)(DT)(DT)(DA)(DT)(DG)(DG)(DC) (DC) (DT)(DG)(DC)(DA)

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 630357
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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