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- PDB-9lp9: The cryo-EM structure of retron Eco8 in a standby state -

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Basic information

Entry
Database: PDB / ID: 9lp9
TitleThe cryo-EM structure of retron Eco8 in a standby state
Components
  • DNA (75-MER)
  • RNA (161-MER)
  • Retron Eco8 OLD nuclease
  • Retron Eco8 reverse transcriptase
KeywordsANTITOXIN/DNA/RNA / retron / multi-copy single-stranded DNA / Eco8 / anti-phage defense / OLD-family endonuclease / reverse transcriptase / 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
: / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Retron Eco8 OLD nuclease / Retron Eco8 reverse transcriptase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYuan, L. / Feng, Y.
Funding support China, 1items
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, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Retron Eco8 OLD nuclease
A: Retron Eco8 reverse transcriptase
C: RNA (161-MER)
D: DNA (75-MER)
E: Retron Eco8 OLD nuclease
I: Retron Eco8 OLD nuclease
M: Retron Eco8 OLD nuclease
H: DNA (75-MER)
L: DNA (75-MER)
P: DNA (75-MER)
F: Retron Eco8 reverse transcriptase
J: Retron Eco8 reverse transcriptase
N: Retron Eco8 reverse transcriptase
G: RNA (161-MER)
K: RNA (161-MER)
O: RNA (161-MER)


Theoretical massNumber of molelcules
Total (without water)821,91716
Polymers821,91716
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Retron Eco8 OLD nuclease


Mass: 87373.789 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: old, ERS139198_01420, Ga0119705_103344 / Production host: Escherichia coli (E. coli)
References: UniProt: P0DV58, Hydrolases; Acting on ester bonds
#2: Protein
Retron Eco8 reverse transcriptase / RT


Mass: 43273.203 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ret, ERS139198_01421, Ga0119705_103345 / Production host: Escherichia coli (E. coli) / References: UniProt: P0DV59, RNA-directed DNA polymerase
#3: RNA chain
RNA (161-MER)


Mass: 51705.531 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: GenBank: 1881611662
#4: DNA chain
DNA (75-MER)


Mass: 23126.848 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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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: retron Eco8 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI MORGAGNI
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: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2PHENIX1.20.1_4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34883 / Symmetry type: POINT
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00848120
ELECTRON MICROSCOPYf_angle_d0.93867012
ELECTRON MICROSCOPYf_dihedral_angle_d21.0989992
ELECTRON MICROSCOPYf_chiral_restr0.057692
ELECTRON MICROSCOPYf_plane_restr0.0066764

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