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- EMDB-61577: Cryo-EM structure of DRT2-RT-ncRNA binary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-61577
TitleCryo-EM structure of DRT2-RT-ncRNA binary complex
Map data
Sample
  • Complex: a complex
    • Other: nucleic acid
    • Protein or peptide: Antiviral reverse transcriptase Drt2
  • Ligand: MAGNESIUM ION
Keywordsa complex / ANTIVIRAL PROTEIN
Function / homology: / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / RNA-directed DNA polymerase activity / DNA/RNA polymerase superfamily / Antiviral reverse transcriptase Drt2
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsZhang H / Zhang S
Funding support China, 1 items
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, 2024-
Header (metadata) releaseApr 22, 2026-
Map releaseApr 22, 2026-
UpdateApr 22, 2026-
Current statusApr 22, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61577.png

Downloads & links

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Map

FileDownload / File: emd_61577.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.08556708 - 0.2701268
Average (Standard dev.)0.0011497004 (±0.012406004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_61577_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_61577_half_map_2.map
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Sample components

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Entire : a complex

EntireName: a complex
Components
  • Complex: a complex
    • Other: nucleic acid
    • Protein or peptide: Antiviral reverse transcriptase Drt2
  • Ligand: MAGNESIUM ION

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Supramolecule #1: a complex

SupramoleculeName: a complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Macromolecule #1: Antiviral reverse transcriptase Drt2

MacromoleculeName: Antiviral reverse transcriptase Drt2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 49.788645 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

UniProtKB: Antiviral reverse transcriptase Drt2

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Macromolecule #2: nucleic acid

MacromoleculeName: nucleic acid / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 91.534867 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(DG)(DA)(DT)(DA)(DT)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / 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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING ONLY
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 347211
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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