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- EMDB-60892: Cryo-EM structure of the large serine recombinase Bxb1 in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-60892
TitleCryo-EM structure of the large serine recombinase Bxb1 in complex with attP and attB (CA/CA CDN) in the intermediate-strand exchange state 2
Map datadeepemhancer
Sample
  • Complex: The large serine recombinase Bxb1 in complex with attP and attB (CA/CA CDN) in the intermediate-strand exchange state 2
    • Complex: large serine recombinase
      • Protein or peptide: Integrase
    • Complex: attP
      • DNA: attP-L
      • DNA: attP-L
      • DNA: attP-R
      • DNA: attP-R
    • Complex: attB
      • DNA: attB-L
      • DNA: attB-L
      • DNA: attB-R
      • DNA: attB-R
  • Ligand: ZINC ION
Keywordslarge serine recombinase / DNA BINDING PROTEIN
Function / homology
Function and homology information


DNA strand exchange activity / DNA binding
Similarity search - Function
Recombinase / DNA-binding recombinase domain / DNA-binding recombinase domain superfamily / DNA-binding recombinase domain profile. / Resolvase/invertase-type recombinase catalytic domain profile. / : / Resolvase, N-terminal catalytic domain / Resolvase-like, N-terminal catalytic domain superfamily / Resolvase, N terminal domain / Resolvase, N terminal domain
Similarity search - Domain/homology
Biological speciesMycobacterium phage Bxb1 (virus) / Mycolicibacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.08 Å
AuthorsSoma T / Hiraizumi M / Yamashita K / Nishimasu H
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJAX232F Japan
Japan Science and TechnologyJPMJCR23B6 Japan
CitationJournal: Mol Cell / Year: 2026
Title: Structure and engineering of the large serine recombinase Bxb1 for gene integration.
Authors: Teppei Soma / Masahiro Hiraizumi / Christopher W Fell / Dario Tagliaferri / Jason Lequyer / Sae Okazaki / Yukari Isayama / Kazuki Kato / Sworaj Sapkota / Harsh Ramani / Benjamin Arya / Cian ...Authors: Teppei Soma / Masahiro Hiraizumi / Christopher W Fell / Dario Tagliaferri / Jason Lequyer / Sae Okazaki / Yukari Isayama / Kazuki Kato / Sworaj Sapkota / Harsh Ramani / Benjamin Arya / Cian Schmitt-Ulms / Keitaro Yamashita / Jonathan S Gootenberg / Omar O Abudayyeh / Hiroshi Nishimasu /
Abstract: The large serine recombinase Bxb1 catalyzes recombination between DNA molecules containing compatible attP and attB sequences, offering broad applications in genome engineering and gene therapies. ...The large serine recombinase Bxb1 catalyzes recombination between DNA molecules containing compatible attP and attB sequences, offering broad applications in genome engineering and gene therapies. Here, we present cryo-electron microscopy structures of the Bxb1-attP-attB synaptic complex in four distinct functional states during its recombination cycle. Notably, the Bxb1 complex structures in the pre-, mid-, and post-strand-exchange states explain how the attP- and attB-bound Bxb1 dimers are assembled into a tetrameric synaptic complex and how an approximately 180° rotation occurs between the left and right dimers after DNA cleavage, thereby enabling DNA strand exchange and religation. Furthermore, we engineered Bxb1 variants with altered DNA preferences and enhanced recombination activity, which improved programmable gene integration in human cells. Overall, our findings advance the mechanistic understanding of large serine recombinases and provide a structural framework for future engineering of Bxb1-mediated genome integration technologies.
History
DepositionJul 20, 2024-
Header (metadata) releaseMar 4, 2026-
Map releaseMar 4, 2026-
UpdateJun 24, 2026-
Current statusJun 24, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_60892.png

Downloads & links

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Map

FileDownload / File: emd_60892.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdeepemhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.41 Å/pix.
x 200 pix.
= 282.2 Å		1.41 Å/pix.
x 200 pix.
= 282.2 Å		1.41 Å/pix.
x 200 pix.
= 282.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.411 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0017584201 - 2.0232143
Average (Standard dev.)0.0015049205 (±0.025959423)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 282.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_60892_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_60892_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_60892_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The large serine recombinase Bxb1 in complex with attP and attB (...

EntireName: The large serine recombinase Bxb1 in complex with attP and attB (CA/CA CDN) in the intermediate-strand exchange state 2
Components
  • Complex: The large serine recombinase Bxb1 in complex with attP and attB (CA/CA CDN) in the intermediate-strand exchange state 2
    • Complex: large serine recombinase
      • Protein or peptide: Integrase
    • Complex: attP
      • DNA: attP-L
      • DNA: attP-L
      • DNA: attP-R
      • DNA: attP-R
    • Complex: attB
      • DNA: attB-L
      • DNA: attB-L
      • DNA: attB-R
      • DNA: attB-R
  • Ligand: ZINC ION

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Supramolecule #1: The large serine recombinase Bxb1 in complex with attP and attB (...

SupramoleculeName: The large serine recombinase Bxb1 in complex with attP and attB (CA/CA CDN) in the intermediate-strand exchange state 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2, #4, #3, #5-#6, #8, #7, #9

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Supramolecule #2: large serine recombinase

SupramoleculeName: large serine recombinase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)

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Supramolecule #3: attP

SupramoleculeName: attP / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2, #4, #3, #5
Source (natural)Organism: Mycobacterium phage Bxb1 (virus) / Synthetically produced: Yes

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Supramolecule #4: attB

SupramoleculeName: attB / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6, #8, #7, #9
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria) / Synthetically produced: Yes

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)
Molecular weightTheoretical: 56.439949 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRALVVIRLS RVTDATTSPE RQLESCQQLC AQRGWDVVGV AEDLDVSGAV DPFDRKRRPN LARWLAFEEQ PFDVIVAYRV DRLTRSIRH LQQLVHWAED HKKLVVSATE AHFDTTTPFA AVVIALMGTV AQMELEAIKE RNRSAAHFNI RAGKYRGSLP P WGYLPTRV ...String:
MRALVVIRLS RVTDATTSPE RQLESCQQLC AQRGWDVVGV AEDLDVSGAV DPFDRKRRPN LARWLAFEEQ PFDVIVAYRV DRLTRSIRH LQQLVHWAED HKKLVVSATE AHFDTTTPFA AVVIALMGTV AQMELEAIKE RNRSAAHFNI RAGKYRGSLP P WGYLPTRV DGEWRLVPDP VQRERILEVY HRVVDNHEPL HLVAHDLNRR GVLSPKDYFA QLQGREPQGR EWSATALKRS MI SEAMLGY ATLNGKTVRD DDGAPLVRAE PILTREQLEA LRAELVKTSR AKPAVSTPSL LLRVLFCAVC GEPAYKFAGG GRK HPRYRC RSMGFPKHCG NGTVAMAEWD AFCEEQVLDL LGDAERLEKV WVAGSDSAVE LAEVNAELVD LTSLIGSPAY RAGS PQREA LDARIAALAA RQEELEGLEA RPSGWEWRET GQRFGDWWRE QDTAAKNTWL RSMNVRLTFD VRGGLTRTID FGDLQ EYEQ HLRLGSVVER LHTGMS

UniProtKB: Integrase

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Macromolecule #2: attP-L

MacromoleculeName: attP-L / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)
Molecular weightTheoretical: 8.315329 KDa
SequenceString:
(DG)(DT)(DG)(DG)(DT)(DT)(DT)(DG)(DT)(DC) (DT)(DG)(DG)(DT)(DC)(DA)(DA)(DC)(DC)(DA) (DC)(DC)(DG)(DC)(DG)(DG)(DT)

GENBANK: GENBANK: NC_002656.1

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Macromolecule #3: attP-R

MacromoleculeName: attP-R / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)
Molecular weightTheoretical: 7.667961 KDa
SequenceString:
(DC)(DT)(DC)(DA)(DG)(DT)(DG)(DG)(DT)(DG) (DT)(DA)(DC)(DG)(DG)(DT)(DA)(DC)(DA)(DA) (DA)(DC)(DC)(DC)(DA)

GENBANK: GENBANK: NC_002656.1

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Macromolecule #4: attP-L

MacromoleculeName: attP-L / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)
Molecular weightTheoretical: 7.677963 KDa
SequenceString:
(DC)(DG)(DC)(DG)(DG)(DT)(DG)(DG)(DT)(DT) (DG)(DA)(DC)(DC)(DA)(DG)(DA)(DC)(DA)(DA) (DA)(DC)(DC)(DA)(DC)

GENBANK: GENBANK: NC_002656.1

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Macromolecule #5: attP-R

MacromoleculeName: attP-R / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycobacterium phage Bxb1 (virus)
Molecular weightTheoretical: 8.292346 KDa
SequenceString:
(DT)(DG)(DG)(DG)(DT)(DT)(DT)(DG)(DT)(DA) (DC)(DC)(DG)(DT)(DA)(DC)(DA)(DC)(DC)(DA) (DC)(DT)(DG)(DA)(DG)(DA)(DC)

GENBANK: GENBANK: NC_002656.1

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Macromolecule #6: attB-L

MacromoleculeName: attB-L / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 7.412754 KDa
SequenceString:
(DG)(DG)(DC)(DC)(DG)(DG)(DC)(DT)(DT)(DG) (DT)(DC)(DG)(DA)(DC)(DG)(DA)(DC)(DG)(DG) (DC)(DG)(DC)(DA)

GENBANK: GENBANK: LN831039.1

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Macromolecule #7: attB-R

MacromoleculeName: attB-R / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 6.704316 KDa
SequenceString:
(DC)(DT)(DC)(DC)(DG)(DT)(DC)(DG)(DT)(DC) (DA)(DG)(DG)(DA)(DT)(DC)(DA)(DT)(DC)(DC) (DG)(DG)

GENBANK: GENBANK: LN831039.1

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Macromolecule #8: attB-L

MacromoleculeName: attB-L / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 6.699307 KDa
SequenceString:
(DC)(DG)(DC)(DC)(DG)(DT)(DC)(DG)(DT)(DC) (DG)(DA)(DC)(DA)(DA)(DG)(DC)(DC)(DG)(DG) (DC)(DC)

GENBANK: GENBANK: LN831039.1

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Macromolecule #9: attB-R

MacromoleculeName: attB-R / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 7.404782 KDa
SequenceString:
(DC)(DC)(DG)(DG)(DA)(DT)(DG)(DA)(DT)(DC) (DC)(DT)(DG)(DA)(DC)(DG)(DA)(DC)(DG)(DG) (DA)(DG)(DA)(DC)

GENBANK: GENBANK: LN831039.1

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 114830
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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