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- PDB-9jl3: Cryo-EM structure of DRT2-RT-ncRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 9jl3
TitleCryo-EM structure of DRT2-RT-ncRNA binary complex
Components
  • Antiviral reverse transcriptase Drt2
  • nucleic acid
KeywordsANTIVIRAL PROTEIN / a complex
Function / homology
Function and homology information


RNA-directed DNA polymerase activity
Similarity search - Function
: / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA/RNA hybrid / DNA/RNA hybrid (> 10) / DNA/RNA hybrid (> 100) / Antiviral reverse transcriptase Drt2
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsZhang, H. / Zhang, S.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Molecular mechanism of the type 2 defense-associated reverse transcriptase.
Authors: Zhikun Liu / Fumeng Liao / Wenqi Wu / Chendi Zhang / Caidie Yue / Aoyan Chen / Shuqin Zhang / Yingcan Liu / Bin Liu / Jie Han / Chuyun Zhang / Xiaoshen Wang / Xuzichao Li / Zhuang Li / Heng Zhang / Hang Yin /
Abstract: Defense-associated reverse transcriptase (DRT) systems play a crucial role in prokaryotic defense mechanisms against phage infections. Among the DRT family, DRT2, DRT3, and DRT9 systems employ ...Defense-associated reverse transcriptase (DRT) systems play a crucial role in prokaryotic defense mechanisms against phage infections. Among the DRT family, DRT2, DRT3, and DRT9 systems employ protein-noncoding RNA (ncRNA) co-regulatory mechanisms to execute defense functions. Here, we focus on the DRT2 system from Klebsiella pneumoniae, which consists of a reverse transcriptase (RT) and an essential ncRNA component. Using biochemical and structural approaches, we determine the structure of the DRT2 system and reveal detailed interaction modes between the DRT2-RT protein and the ncRNA, especially mediated by specialized anchoring loops and pseudoknot-related structures. The RT protein adopts a conventional "right-hand" fold, while a flexible region of the ncRNA exhibits dynamic conformations, likely serving as the template for reverse transcription. DRT2 mediates reverse transcription through a conserved DDD catalytic triad that coordinates a divalent Mg²⁺ ion. Notably, a short DNA primer-ncRNA duplex is accommodated in a positively charged pocket formed by the thumb and fingers domains, and both interaction analysis and mutagenesis studies confirm that duplex stabilization is essential for activity. Structural comparison and phylogenetic studies of DRT2 and other RT proteins, such as group II introns and UG/Abi RTs, highlight the unique adaptation with a straight extended thumb domain and specialized structures for ncRNA-binding, exemplifying an evolutionary trajectory of RT proteins. In conclusion, our findings expand the understanding of the distinctive characteristics of the DRT2 system and the diversity of prokaryotic antiviral strategies.
History
DepositionSep 17, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 22, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 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
A: Antiviral reverse transcriptase Drt2
B: nucleic acid
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,3483
Polymers141,3242
Non-polymers241
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: Protein Antiviral reverse transcriptase Drt2 / Reverse transcriptase/maturase family protein


Mass: 49788.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: drt2, AAFQ03_23310, BCV49_04045, QDV38_25290 / Production host: Escherichia coli (E. coli) / References: UniProt: A0ABF7SXL6
#2: DNA/RNA hybrid nucleic acid


Mass: 91534.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli (E. coli)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / 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: a complex / Type: COMPLEX / Entity ID: #2, #1 / Source: RECOMBINANT
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 347211 / Symmetry type: POINT
RefinementHighest resolution: 3.49 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0077693
ELECTRON MICROSCOPYf_angle_d0.76211197
ELECTRON MICROSCOPYf_dihedral_angle_d18.7712527
ELECTRON MICROSCOPYf_chiral_restr0.0421372
ELECTRON MICROSCOPYf_plane_restr0.004782

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