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- PDB-9nlx: Cryo-EM structure of the trimeric SenDRT9 RT-ncRNA complex (GST f... -

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Basic information

Entry
Database: PDB / ID: 9nlx
TitleCryo-EM structure of the trimeric SenDRT9 RT-ncRNA complex (GST fusion)
Components
  • RNA (141-MER)
  • Sen DRT9 reverse transcriptase
KeywordsIMMUNE SYSTEM / DRT9 / Complex
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesSalmonella enterica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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. Defense-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. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other related DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-EM structures reveal how a noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Remarkably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that likely prime DNA synthesis, leading to the formation of high-molecular weight 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 work unveils a novel nucleic acid-driven defense 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
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Revision 1.0May 14, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sen DRT9 reverse transcriptase
G: RNA (141-MER)
B: Sen DRT9 reverse transcriptase
H: RNA (141-MER)
C: Sen DRT9 reverse transcriptase
I: RNA (141-MER)


Theoretical massNumber of molelcules
Total (without water)311,0366
Polymers311,0366
Non-polymers00
Water00
1


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

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Components

#1: Protein Sen DRT9 reverse transcriptase


Mass: 58318.961 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica (bacteria) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): AI
#2: RNA chain RNA (141-MER)


Mass: 45359.656 Da / Num. of mol.: 3
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: Cryo-EM structure of the SenDRT9-encoded RT-ncRNA trimeric complex fused to GST
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
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: 59.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: v4.6.2 / Category: 3D reconstruction / Details: Non-Uniform Refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 367640 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT

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