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- PDB-9nlv: Cryo-EM structure of hexameric SenDRT9 RT-ncRNA complex -

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Basic information

Entry
Database: PDB / ID: 9nlv
TitleCryo-EM structure of hexameric SenDRT9 RT-ncRNA complex
Components
  • RNA (141-MER)
  • RNA-dependent DNA polymerase
KeywordsIMMUNE SYSTEM / DRT9 / Polymerase
Function / homology: / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / RNA / RNA (> 10) / RNA (> 100) / RNA-dependent DNA polymerase
Function and homology information
Biological speciesEscherichia coli (E. coli)
Salmonella enterica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsBurman, N. / Pandey, S. / Wiedenheft, B. / Sternberg, S.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM134867 United States
National Science Foundation (NSF, United States)2239685 United States
CitationJournal: Nature / Year: 2025
Title: Protein-primed homopolymer synthesis by an antiviral reverse transcriptase.
Authors: Stephen Tang / Rimantė Žedaveinytė / Nathaniel Burman / Shishir Pandey / Josephine L Ramirez / Louie M Kulber / Tanner Wiegand / Royce A Wilkinson / Yanzhe Ma / Dennis J Zhang / George D ...Authors: Stephen Tang / Rimantė Žedaveinytė / Nathaniel Burman / Shishir Pandey / Josephine L Ramirez / Louie M Kulber / Tanner Wiegand / Royce A Wilkinson / Yanzhe Ma / Dennis J Zhang / George D Lampe / Mirela Berisa / Marko Jovanovic / Blake Wiedenheft / Samuel H Sternberg /
Abstract: Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation. Defence-associated reverse transcriptase (DRT) systems provide an ...Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein-DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.
History
DepositionMar 3, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 14, 2025Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 4, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Jun 11, 2025Group: Data collection / Database references / Category: citation / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-dependent DNA polymerase
G: RNA (141-MER)
B: RNA-dependent DNA polymerase
C: RNA-dependent DNA polymerase
D: RNA-dependent DNA polymerase
E: RNA-dependent DNA polymerase
F: RNA-dependent DNA polymerase
H: RNA (141-MER)
I: RNA (141-MER)
J: RNA (141-MER)
K: RNA (141-MER)
L: RNA (141-MER)


Theoretical massNumber of molelcules
Total (without water)665,95812
Polymers665,95812
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RNA-dependent DNA polymerase


Mass: 58318.961 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: GQA06_05700 / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI / References: UniProt: A0A6D0I497
#2: RNA chain
RNA (141-MER)


Mass: 52673.969 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica (bacteria) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI
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: Sen DRT9 hexameric ribonucleoprotein complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Salmonella enterica (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: AI
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4.6.2particle selectionBlob Picker
2SerialEMimage acquisition
4cryoSPARCv4.6.2CTF correctionPatch-CTF
7UCSF ChimeraX1.9model fitting
9cryoSPARCv4.6.2initial Euler assignment
10cryoSPARCv4.6.2final Euler assignment
11cryoSPARCv4.6.2classification
12cryoSPARCv4.6.23D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6029711
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500162 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 101.4 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingDetails: The initial model for the RT was produced using AlphaFold 3
Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00343344
ELECTRON MICROSCOPYf_angle_d0.68862784
ELECTRON MICROSCOPYf_dihedral_angle_d26.23515372
ELECTRON MICROSCOPYf_chiral_restr0.0487734
ELECTRON MICROSCOPYf_plane_restr0.0064842

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