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- EMDB-63269: The cryo-EM structure of retron Eco8 in an active state -

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Basic information

Entry
Database: EMDB / ID: EMD-63269
TitleThe cryo-EM structure of retron Eco8 in an active state
Map data
Sample
  • Complex: retron Eco8
    • Protein or peptide: Retron Eco8 OLD nuclease
    • Protein or peptide: Retron Eco8 reverse transcriptase
    • RNA: RNA (161-MER)
    • DNA: DNA (75-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Keywordsretron / multi-copy single-stranded DNA / Eco8 / anti-phage defense / OLD-family endonuclease / reverse transcriptase / ANTITOXIN/DNA/RNA / ANTITOXIN-DNA-RNA complex
Function / homology
Function and homology information


nuclease activity / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / defense response to virus / Hydrolases; Acting on ester bonds / ATP hydrolysis activity / RNA binding / ATP binding / metal ion binding
Similarity search - Function
AAA domain, putative AbiEii toxin, Type IV TA system / : / RNA-directed DNA polymerase (reverse transcriptase), msDNA / : / ATPase, AAA-type, core / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Retron Eco8 OLD nuclease / Retron Eco8 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsYuan L / Feng Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, China) China
CitationJournal: Mol Cell / Year: 2025
Title: Molecular mechanism of Eco8-mediated anti-phage defense.
Authors: Linggang Yuan / Liqiao Xu / Bing Wu / Qingyang Liu / Yue Yao / Xiaoting Hua / Yu Feng /
Abstract: Escherichia coli Eco8 is an anti-phage defense system consisting of a reverse transcriptase, a class 3 overcoming lysogenization defect (OLD) nuclease, and a DNA-RNA chimera called multi-copy single- ...Escherichia coli Eco8 is an anti-phage defense system consisting of a reverse transcriptase, a class 3 overcoming lysogenization defect (OLD) nuclease, and a DNA-RNA chimera called multi-copy single-stranded DNA (msDNA). Genetic and biochemical data suggest that Eco8-mediated anti-phage defense is triggered by the phage single-stranded DNA (ssDNA)-binding proteins, but the underlying structural basis remains unknown. Here, we demonstrate that the DNA cleavage and ATP hydrolysis activities of the OLD nuclease are critical for Eco8-mediated anti-phage defense. We also determine the cryoelectron microscopy (cryo-EM) structures of Eco8 alone and in complex with the T7 phage ssDNA-binding protein. Structural analysis reveals that the reverse transcriptase, msDNA, and OLD nuclease form a megacomplex with a 4:4:4 stoichiometry. The T7 phage ssDNA-binding protein unwinds the msDNA and transforms Eco8 into an ATP-dependent DNA-degrading machinery. This study not only elucidates the molecular mechanism of Eco8-mediated anti-phage defense but also validates that msDNA serves as a sensor of phage DNA-modifying/binding proteins.
History
DepositionJan 24, 2025-
Header (metadata) releaseNov 19, 2025-
Map releaseNov 19, 2025-
UpdateNov 19, 2025-
Current statusNov 19, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63269.png

Downloads & links

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Map

FileDownload / File: emd_63269.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 300 pix.
= 360. Å		1.2 Å/pix.
x 300 pix.
= 360. Å		1.2 Å/pix.
x 300 pix.
= 360. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4284557 - 2.7652922
Average (Standard dev.)0.0042271405 (±0.10011254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 360.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_63269_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63269_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : retron Eco8

EntireName: retron Eco8
Components
  • Complex: retron Eco8
    • Protein or peptide: Retron Eco8 OLD nuclease
    • Protein or peptide: Retron Eco8 reverse transcriptase
    • RNA: RNA (161-MER)
    • DNA: DNA (75-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: retron Eco8

SupramoleculeName: retron Eco8 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Retron Eco8 OLD nuclease

MacromoleculeName: Retron Eco8 OLD nuclease / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 87.373789 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTIESIRVKN LLSFDDVILR DFRDINCIIG RNNVGKSNLL KVIRYFYAKL ENKKVIPLDF HTNYNAVGEI TFTFDTTRIK KIVTSRKNN GRFHKHIYNT LFKSSSVKLN FEELIARKNS TNKSFFSLTL TICKDDSVMW SVDDPKVRSL LATLYPFLYI E TRHIDLYD ...String:
MTIESIRVKN LLSFDDVILR DFRDINCIIG RNNVGKSNLL KVIRYFYAKL ENKKVIPLDF HTNYNAVGEI TFTFDTTRIK KIVTSRKNN GRFHKHIYNT LFKSSSVKLN FEELIARKNS TNKSFFSLTL TICKDDSVMW SVDDPKVRSL LATLYPFLYI E TRHIDLYD WNPIWKLISN LNSFNFDDVD HDELVNFLDE KISSRKGDYK KYIDRVVSVI DTKPYTYKEK VINYIKVAIK GD SFVNAGE ELFTQSDGTN SNKFLETLLH LLITLTRTEF ISPIVYIDEP EVGLHPKLAE SFVSNLNKIY SKFKKTSELS GPG RYKTPY PNIFYSTHSP SILKQTIKLF GKDQQVLHFS KKKDGSTRVN KINSTYSDER FLNIFSDNEA RLFFSEYIVF VEGA TELEL FRNLSLLNLY PAFSLADIYD ANEVILANIN PGYSKASIPF VIIKDIDTLI DYSIKTEKFS LRPLFEKMIK ELTKE FDYY DTGFGRVRKE IDLFSDIQSS TKKHMDSGLF FKRFSLHNLS SRINKVSRKL NRYFMTTTIE GALINEQSLP YFFNWI GDV ILTQMTINNP NPDKFIEAMR RRYNIKSQVV PLFKSVFCIG LNHPVYSSAV DKQALRIKLS FLNYLKRKVY SDFNNEK EI VLALRLAFGG KTETQYTLDK LRKDGEAELF REKIKNYKNN ELFFLEPQMT KTSGWVTTFL NYTIEKITSE ESDDDRIR Q KLSFIFPEII SIIEQASSSI EAEESSLTG

UniProtKB: Retron Eco8 OLD nuclease

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Macromolecule #2: Retron Eco8 reverse transcriptase

MacromoleculeName: Retron Eco8 reverse transcriptase / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 43.273203 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKTKKMILVD KVFYEKILSV ESFKENIITQ SAIPKISNKE VRLISSGSKI FYAINNTSPH SHVQLRLNRF FLSHIPLNSA AKAFVRGGS YLKYLEPHIY GSSYCRLDIS SFFNNISFDD VKQSLSPYIK DEYLIGTEQK LIDAILNSVG YESPIRKDKG M IIPMGFRT ...String:
MKTKKMILVD KVFYEKILSV ESFKENIITQ SAIPKISNKE VRLISSGSKI FYAINNTSPH SHVQLRLNRF FLSHIPLNSA AKAFVRGGS YLKYLEPHIY GSSYCRLDIS SFFNNISFDD VKQSLSPYIK DEYLIGTEQK LIDAILNSVG YESPIRKDKG M IIPMGFRT SPAISNIVFR KMDLLIQDFC AKKGVIYSRY ADDMLFSNPR ESKLLMSDYF IDEISSLLSI MGFNINQSKY IS REKEISI NGYVIENKGG NGSIGTIRLS KSKLNTVLKV THALAQNIPY KNICNKYIKV RLKEKNIKYE SKKDEFEKKY YRD QLINYL GGYRSYLISL VKFHSEYKCV NSDFIIQING ILNDIQNHIQ KIKKNRRL

UniProtKB: Retron Eco8 reverse transcriptase

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Macromolecule #3: RNA (161-MER)

MacromoleculeName: RNA (161-MER) / type: rna / ID: 3 / Number of copies: 4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 51.705531 KDa
SequenceString:
GCUUCUUCUU CGAUAGAAGC UGAGGAGUCC AGUUUGACUG GAUAAGGUGU UCGCCAUCUC UAGCCUCAGU AAAAACUAGC UCAUCCUUU GCGCACUUGG CGCUAUAGGU AACUUUAAUA CUCACAGAGU GUAAAGAUUG GUCUUGGUAU CGGAGAAGAA G C

GENBANK: GENBANK: CP057441.1

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Macromolecule #4: DNA (75-MER)

MacromoleculeName: DNA (75-MER) / type: dna / ID: 4 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.126848 KDa
SequenceString: (DC)(DA)(DA)(DG)(DA)(DC)(DC)(DA)(DA)(DT) (DC)(DT)(DT)(DT)(DA)(DC)(DA)(DC)(DT)(DC) (DT)(DG)(DT)(DG)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DA)(DG)(DT)(DT)(DA)(DC)(DC) (DT) (DA)(DT)(DA)(DG)(DC)(DG) ...String:
(DC)(DA)(DA)(DG)(DA)(DC)(DC)(DA)(DA)(DT) (DC)(DT)(DT)(DT)(DA)(DC)(DA)(DC)(DT)(DC) (DT)(DG)(DT)(DG)(DA)(DG)(DT)(DA)(DT) (DT)(DA)(DA)(DA)(DG)(DT)(DT)(DA)(DC)(DC) (DT) (DA)(DT)(DA)(DG)(DC)(DG)(DC)(DC) (DA)(DA)(DG)(DT)(DG)(DC)(DG)(DC)(DA)(DA) (DA)(DG) (DG)(DA)(DT)(DG)(DA)(DG)(DC) (DT)(DA)(DG)(DT)(DT)(DT)(DT)(DT)

GENBANK: GENBANK: CP057441.1

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 4 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI MORGAGNI
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 25017
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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