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- EMDB-64992: Cryo-EM structure of the catalytically inactive DRT4 -

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Basic information

Entry
Database: EMDB / ID: EMD-64992
TitleCryo-EM structure of the catalytically inactive DRT4
Map data
Sample
  • Complex: DRT4
    • Protein or peptide: Reverse transcriptase domain-containing protein
KeywordsUG15 / DRT4 / Reverse transcriptases / UG/Abi system / DNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA complex
Function / homologyReverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase domain-containing protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsWang Y / Deng Z
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Anti-phage defense mechanism involving phage-encoded DNA binding protein and bacterial reverse transcriptase DRT4.
Authors: Yong Wang / Hao Wu / Jinyue Li / Qiyin An / Zhenhua Tian / Zengqin Deng /
Abstract: Prokaryotic defense-associated reverse transcriptase (DRT) systems confer host resistance to viral infection through DNA synthesis; however, the molecular mechanisms underlying their function remain ...Prokaryotic defense-associated reverse transcriptase (DRT) systems confer host resistance to viral infection through DNA synthesis; however, the molecular mechanisms underlying their function remain poorly understood. Here, we demonstrate that DRT4, a single-gene anti-phage defense system, synthesizes single-stranded DNA (ssDNA) products of random sequences in a template-independent manner. High-resolution cryo-EM structures of DRT4 in multiple functional states elucidate its oligomeric architecture, catalytic metal ion coordination, and substrate/DNA product binding, offering mechanistic insights into its promiscuous polymerization activity. Structural and biochemical analyses further identify a conserved tyrosine residue that acts as the priming site for the initiation of DNA synthesis. Upon phage infection, a phage-encoded DNA-binding protein, ORF55, protects the 3' end of the DRT4-synthesized ssDNA from host exonuclease degradation, likely resulting in toxic ssDNA accumulation that leads to cell death. Remarkably, ORF55 also activates DRT6, a structural homolog of DRT4, suggesting a conserved activation mechanism among related DRT systems. These findings provide structural and mechanistic insights into DRT4-mediated antiviral defense, establishing a distinct paradigm for antiviral reverse transcriptase in bacterial immunity.
History
DepositionJun 9, 2025-
Header (metadata) releaseNov 12, 2025-
Map releaseNov 12, 2025-
UpdateJan 21, 2026-
Current statusJan 21, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_64992.png

Downloads & links

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Map

FileDownload / File: emd_64992.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.)
0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-1.3607749 - 2.143959
Average (Standard dev.)0.00032217804 (±0.061832614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 285.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64992_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64992_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : DRT4

EntireName: DRT4
Components
  • Complex: DRT4
    • Protein or peptide: Reverse transcriptase domain-containing protein

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Supramolecule #1: DRT4

SupramoleculeName: DRT4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Reverse transcriptase domain-containing protein

MacromoleculeName: Reverse transcriptase domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 63.823031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HHHHHHSSGM VIFDEKRHLY EALLRHNYFP NQKGSISEIP PCFSSRTFTP EIAELISSDT SGRRSLQGYD CVEYYATRYN NFPRTLSII HPKAYSKLAK HIHDNWEEIR FIKENENSMI KPDMHADGRI IIMNYEDAET KTIRELNDGF GRRFKVNADI S GCFTNIYS ...String:
HHHHHHSSGM VIFDEKRHLY EALLRHNYFP NQKGSISEIP PCFSSRTFTP EIAELISSDT SGRRSLQGYD CVEYYATRYN NFPRTLSII HPKAYSKLAK HIHDNWEEIR FIKENENSMI KPDMHADGRI IIMNYEDAET KTIRELNDGF GRRFKVNADI S GCFTNIYS HSIPWAVIGV NNAKIALNTK VKNQDKHWSD KLDYFQRQAK RNETHGVPIG PATSSIVCEI ILSAVDKRLR DD GFLFRRY IAAYTCYCKT HDDAKEFLHL LGMELSKYKL SLNLHKTKIT NLPGTLNDNW VSLLNVNSPT KKRFTDQDLN KLS SSEVIN FLDYAVQLNT QVGGGSILKY AISLVINNLD EYTITQVYDY LLNLSWHYPM LIPYLGVLIE HVYLDDGDEY KNKF NEILS MCAENKCSDG MAWTLYFCIK NNIDIDDDVI EKIICFGDCL SLCLLDSSDI YEEKINNFVS DIIKLDYEYD IDRYW LLFY QRFFKDKAPS PYNDKCFDIM KGYGVDFMPD ENYKTKAESY CHVVNNPFLE DGDEIVSFND YMAIA

UniProtKB: Reverse transcriptase domain-containing protein

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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

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Coot / Number images used: 126192
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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