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- PDB-9ljf: Cryo-EM structure of the dGTP bound DRT2-ncRNA complex -

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Basic information

Entry
Database: PDB / ID: 9ljf
TitleCryo-EM structure of the dGTP bound DRT2-ncRNA complex
Components
  • DNA (5'-D(P*GP*AP*TP*AP*T)-3')
  • RNA (280-MER)
  • RNA-dependent DNA polymerase
KeywordsANTIVIRAL PROTEIN/RNA/DNA / ANTIVIRAL PROTEIN-RNA-DNA complex
Function / homology2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / : / DNA / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsZhang, J.T. / Qing, H. / Xia, Y.S. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Host Microbe / Year: 2026
Title: Dual sensing activates antiviral reverse transcriptase for membrane targeting.
Authors: Yushan Xia / Hua Qi / Xin-Yang Wei / Jun-Tao Zhang / Wenao Hu / Yu-Jun Liu / Xin-Yi Song / Xue Liu / Chongyuan Wang / Ning Jia /
Abstract: The prokaryotic type 2 defense-associated reverse transcriptase (DRT2) system mediates antiviral defense by catalyzing the rolling-circle reverse transcription of a noncoding RNA (ncRNA) and ...The prokaryotic type 2 defense-associated reverse transcriptase (DRT2) system mediates antiviral defense by catalyzing the rolling-circle reverse transcription of a noncoding RNA (ncRNA) and producing the toxic Neo protein that arrests bacterial growth. However, the mechanisms underlying DRT2 activation and effector function remain unknown. Here, we identified two distinct activation mechanisms: direct binding of a phage-encoded single-stranded DNA-binding protein (SSB or SSAP), and the detection of elevated intracellular dGTP levels induced by the phage-encoded ribonucleotide reductase NrdAB. Upon activation, the produced Neo protein directly targets the bacterial membrane, inducing membrane depolarization and growth arrest. Cryo-electron microscopy (cryo-EM) structures of the DRT2-ncRNA complex in its arrested and dGTP-bound active states provide mechanistic insights into rolling-circle ccDNA synthesis and template jumping. Furthermore, these identified activation mechanisms enable the DRT2 system with an engineered ncRNA template to produce a large-scale, user-defined double-stranded DNA (dsDNA) template in vivo, highlighting its potential in biotechnological applications.
History
DepositionJan 15, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 15, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-dependent DNA polymerase
B: RNA (280-MER)
C: DNA (5'-D(P*GP*AP*TP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,8346
Polymers141,2793
Non-polymers5563
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 RNA-dependent DNA polymerase


Mass: 49788.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: RNA chain RNA (280-MER)


Mass: 89970.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: GenBank: 1920786575
#3: DNA chain DNA (5'-D(P*GP*AP*TP*AP*T)-3')


Mass: 1519.048 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-DGT / 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: dGTP bound DRT2-ncRNA complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150565 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047148
ELECTRON MICROSCOPYf_angle_d0.87210369
ELECTRON MICROSCOPYf_dihedral_angle_d19.2492260
ELECTRON MICROSCOPYf_chiral_restr0.0411255
ELECTRON MICROSCOPYf_plane_restr0.007749

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