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- PDB-9i2f: Cryo-EM structure of retron Eco2 (Ec67) -

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Basic information

Entry
Database: PDB / ID: 9i2f
TitleCryo-EM structure of 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: 2.8 Å
AuthorsJasnauskaite, M. / Miksys, A. / Skorupskaite, A. / Malinauskaite, L. / Pausch, P.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)5342-2023European Union
CitationJournal: To Be Published
Title: Structure and mechanism of anti-phage retron Eco2
Authors: Jasnauskaite, M. / Juozapaitis, J. / Liegute, T. / Grigaitis, R. / Skorupskaite, A. / Steinchen, W. / Miksys, M. / Truncaite, L. / Kazlauskaite, K. / Torres Jimenez, M.F. / Khochare, S. / ...Authors: Jasnauskaite, M. / Juozapaitis, J. / Liegute, T. / Grigaitis, R. / Skorupskaite, A. / Steinchen, W. / Miksys, M. / Truncaite, L. / Kazlauskaite, K. / Torres Jimenez, M.F. / Khochare, S. / Dudas, G. / Bange, G. / Malinauskaite, L. / Songailiene, I. / Pausch, P.
History
DepositionJan 20, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)394,8829
Polymers394,8829
Non-polymers00
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: 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)
#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: 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)
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 retron Eco2 (Ec67) with RNA and DNA
Type: COMPLEX / Entity ID: all / 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
1100 mMTris-HCl1
2150 mMNaCl1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA current / 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: 30.31 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1988
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3particle selection
2PHENIX1.21.2_5419model refinement
3EPU3.2image acquisition
5cryoSPARC4.3CTF correction
13cryoSPARC4.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 544986
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 504525 / Symmetry type: POINT
Atomic model building
ID 3D fitting-IDSource nameType
11AlphaFoldin silico model
21
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320689
ELECTRON MICROSCOPYf_angle_d0.54329108
ELECTRON MICROSCOPYf_dihedral_angle_d21.1795480
ELECTRON MICROSCOPYf_chiral_restr0.0383405
ELECTRON MICROSCOPYf_plane_restr0.0052709

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