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- EMDB-63146: Cryo-EM structure of the DRT2-ncRNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-63146
TitleCryo-EM structure of the DRT2-ncRNA complex
Map data
Sample
  • Complex: DRT2-ncRNA complex
    • Protein or peptide: RNA-dependent DNA polymerase
    • RNA: RNA (280-MER)
    • DNA: DNA (5'-D(P*GP*AP*TP*AP*T)-3')
  • Ligand: MAGNESIUM ION
KeywordsRNA BINDING PROTEIN-RNA-DNA COMPLEX / ANTIVIRAL PROTEIN/RNA/DNA / ANTIVIRAL PROTEIN-RNA-DNA complex
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsZhang JT / Qing H / Xia YS / Song XY / Jia N
Funding support1 items
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, 2025-
Header (metadata) releaseJul 15, 2026-
Map releaseJul 15, 2026-
UpdateSep 16, 2026-
Current statusSep 16, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63146.png

Downloads & links

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Map

FileDownload / File: emd_63146.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 248.1 Å		0.83 Å/pix.
x 300 pix.
= 248.1 Å		0.83 Å/pix.
x 300 pix.
= 248.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-2.6784575 - 3.679063
Average (Standard dev.)-0.0003185013 (±0.06268213)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_63146_msk_1.map
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Half map: #2

Fileemd_63146_half_map_1.map
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Half map: #1

Fileemd_63146_half_map_2.map
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Sample components

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Entire : DRT2-ncRNA complex

EntireName: DRT2-ncRNA complex
Components
  • Complex: DRT2-ncRNA complex
    • Protein or peptide: RNA-dependent DNA polymerase
    • RNA: RNA (280-MER)
    • DNA: DNA (5'-D(P*GP*AP*TP*AP*T)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DRT2-ncRNA complex

SupramoleculeName: DRT2-ncRNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Macromolecule #1: RNA-dependent DNA polymerase

MacromoleculeName: RNA-dependent DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 50.890758 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MNNDDYPWFR KRGYLHFDEP VSLKKAVKYV SSPEKIIKHS FLPFLSFEVK SFKIKKDKST KQLSKTEKLR PIAYSSHLDS HIYAFYAEY LTGHYELLIQ ENNLHENILA FRSLNKSNIE FAKRAFDTIT EMGECSAVAL DLSGFFDNLD HQILKHQWCK V IGTEALPQ ...String:
MNNDDYPWFR KRGYLHFDEP VSLKKAVKYV SSPEKIIKHS FLPFLSFEVK SFKIKKDKST KQLSKTEKLR PIAYSSHLDS HIYAFYAEY LTGHYELLIQ ENNLHENILA FRSLNKSNIE FAKRAFDTIT EMGECSAVAL DLSGFFDNLD HQILKHQWCK V IGTEALPQ DHFAIYKSIT RYSKVDKNRA YEILGISKNN PKYNRRKICT PVDFRNKIRK NGLIIVNNSQ KGIPQGSPIS AL LSNIYML DFDIEMRDYA QERGGHYYRY CDDMLFIVPT KYNKTLAGDV AQRIKHLKVE LNTKKTEIRD FIYKDSTLVA NMP LQYLGF IFDGSNILLR SSSLARYSER MKRGVRLAKA TMDSKNRIRE NKGEALKALF KKKLYARYSH IGRRNFLTYG YRAA KIMNS KAIKRQLKPL QKRLENEILK GSHHHHHHE

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Macromolecule #2: RNA (280-MER)

MacromoleculeName: RNA (280-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 89.970867 KDa
SequenceString: GCCCUAAACA AAGGUUUAGG GGUAUUGUAC AGGUUGUCAA GCCUCCCACA GGUCUUGGUG AAACCAAUCA CUGUGACGAC GGUAAGCAA CACUUGGAUG AUAUUCAUAA UUGACUCCAC GCUACUGAUU ACAUUAUACA GCAUAUCUAA CAUUUGCGGC G AGGUUCAC ...String:
GCCCUAAACA AAGGUUUAGG GGUAUUGUAC AGGUUGUCAA GCCUCCCACA GGUCUUGGUG AAACCAAUCA CUGUGACGAC GGUAAGCAA CACUUGGAUG AUAUUCAUAA UUGACUCCAC GCUACUGAUU ACAUUAUACA GCAUAUCUAA CAUUUGCGGC G AGGUUCAC AAUUUGUAUU UAGGUACUGA UUGUGGAUGA GAAGGUUGGA GAAAGACCAC UUGGUUAAGC CGGAGGAUGU GU CCUAGAA UUGUCGCUAU UCUGUCAUCC UCCGGUUUUG CUA

GENBANK: GENBANK: CP063277.1

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Macromolecule #3: DNA (5'-D(P*GP*AP*TP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*GP*AP*TP*AP*T)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 1.519048 KDa
SequenceString:
(DG)(DA)(DT)(DA)(DT)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 415937
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT

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