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- PDB-9s1f: Cryo-EM structure of activated retron Eco2 (Ec67) -

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Basic information

Entry
Database: PDB / ID: 9s1f
TitleCryo-EM structure of activated retron Eco2 (Ec67)
Components
  • RNA (132-MER)
  • Retron Ec67 protein
  • msDNA (67-MER)
KeywordsANTIVIRAL PROTEIN / Retron / Reverse transcriptase / DNA / RNA / TOPRIM / RNaseH / msDNA
Function / homology
Function and homology information


ribonuclease H / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / defense response to virus / RNA binding / metal ion binding
Similarity search - Function
: / RNA-directed DNA polymerase (reverse transcriptase), msDNA / : / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Retron Ec67 protein
Similarity search - Component
Biological speciesEscherichia coli NCTC 86 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSkorupskaite, A. / Jasnauskaite, M. / Grigaitis, R. / Malinauskaite, L. / Pausch, P.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)5342-2023European Union
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Structure and mechanism of antiphage retron Eco2.
Authors: M Jasnauskaitė / J Juozapaitis / T Liegutė / R Grigaitis / A Skorupskaitė / W Steinchen / A Mikšys / L Truncaitė / K Kazlauskaitė / M F Torres Jiménez / S Khochare / G Dudas / G Bange ...Authors: M Jasnauskaitė / J Juozapaitis / T Liegutė / R Grigaitis / A Skorupskaitė / W Steinchen / A Mikšys / L Truncaitė / K Kazlauskaitė / M F Torres Jiménez / S Khochare / G Dudas / G Bange / L Malinauskaitė / I Songailienė / P Pausch /
Abstract: Retrons are prokaryotic reverse transcriptase systems that produce multicopy single-stranded DNA (msDNA), yet the principles by which they mediate antiviral defense remain largely unresolved. Here we ...Retrons are prokaryotic reverse transcriptase systems that produce multicopy single-stranded DNA (msDNA), yet the principles by which they mediate antiviral defense remain largely unresolved. Here we investigate the mechanism of Escherichia coli Eco2, a minimal retron composed of a single reverse transcriptase-nuclease fusion protein. Cryogenic electron microscopy and hydrogen/deuterium exchange mass spectrometry reveal the structures and dynamics of a trimeric nucleoprotein complex assembled within a branched msDNA scaffold, which cages the TOPRIM nucleases. We show that the phage-encoded endonuclease DenB initiates msDNA degradation, thereby unblocking the nuclease active sites. Activated Eco2 cuts transfer RNAs, resulting in translational shutdown for antiphage defense. We further identify ribosomal protein S1 as a putative RNA chaperone that associates with the msDNA precursor. These findings provide insights into the molecular mechanisms of minimal retrons and establish a structural basis for engineering of Eco2.
History
DepositionJul 18, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 18, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: RNA (132-MER)
E: RNA (132-MER)
F: RNA (132-MER)
B: Retron Ec67 protein
G: msDNA (67-MER)
I: msDNA (67-MER)
A: Retron Ec67 protein
H: msDNA (67-MER)
C: Retron Ec67 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)395,05216
Polymers394,8829
Non-polymers1707
Water00
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

#1: RNA chain RNA (132-MER)


Mass: 42593.203 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli NCTC 86 (bacteria) / Strain: 23 / Production host: Escherichia coli DH5[alpha] (bacteria)
#2: Protein Retron Ec67 protein / ORF4-Ec67 RT


Mass: 68511.922 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli NCTC 86 (bacteria) / Strain: 23 / Gene: ret, Ga0175966_113075, RG66_23265 / Production host: Escherichia coli DH5[alpha] (bacteria)
References: UniProt: P21325, RNA-directed DNA polymerase, ribonuclease H
#3: DNA chain msDNA (67-MER)


Mass: 20522.113 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli NCTC 86 (bacteria) / Strain: 23 / Production host: Escherichia coli DH5[alpha] (bacteria)
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
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: Ternary complex of activated retron Eco2 (Ec67) with RNA and DNA
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli NCTC 86 (bacteria) / Strain: 23
Source (recombinant)Organism: Escherichia coli DH5[alpha] (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HCl1
2150 mMNaCl1
310 mMMgCl21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 29 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2968
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7particle selection
2PHENIX1.21.2_5419model refinement
3EPU3.1image acquisition
13cryoSPARC4.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2271864 / Details: Template picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 617364 / Details: Symmetry expanded particles were used / Symmetry type: POINT
Atomic model building
ID 3D fitting-IDSource nameTypeAccession codeDetailsInitial refinement model-ID
11AlphaFoldin silico model
21Otherexperimental modelD_1292144007Model from another deposition2
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319182
ELECTRON MICROSCOPYf_angle_d0.54426823
ELECTRON MICROSCOPYf_dihedral_angle_d18.7594745
ELECTRON MICROSCOPYf_chiral_restr0.0373141
ELECTRON MICROSCOPYf_plane_restr0.0052611

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