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- EMDB-49523: Cryo-EM structure of hexameric SenDRT9 RT-ncRNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-49523
TitleCryo-EM structure of hexameric SenDRT9 RT-ncRNA complex
Map data
Sample
  • Complex: Sen DRT9 hexameric ribonucleoprotein complex
    • Protein or peptide: RNA-dependent DNA polymerase
    • RNA: RNA (141-MER)
KeywordsDRT9 / Polymerase / Immune System
Function / homology: / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / RNA-dependent DNA polymerase
Function and homology information
Biological speciesSalmonella enterica (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsBurman N / Pandey S / Wiedenheft B / Sternberg SH
Funding support United States, 2 items
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, 2025-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateJun 11, 2025-
Current statusJun 11, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49523.png

Downloads & links

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Map

FileDownload / File: emd_49523.map.gz / Format: CCP4 / Size: 465.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 496 pix.
= 449.426 Å		0.91 Å/pix.
x 496 pix.
= 449.426 Å		0.91 Å/pix.
x 496 pix.
= 449.426 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9061 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.2226207 - 0.6894974
Average (Standard dev.)-0.00008720286 (±0.017356757)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions496496496
Spacing496496496
CellA=B=C: 449.4256 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49523_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: #1

Fileemd_49523_half_map_2.map
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Sample components

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Entire : Sen DRT9 hexameric ribonucleoprotein complex

EntireName: Sen DRT9 hexameric ribonucleoprotein complex
Components
  • Complex: Sen DRT9 hexameric ribonucleoprotein complex
    • Protein or peptide: RNA-dependent DNA polymerase
    • RNA: RNA (141-MER)

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Supramolecule #1: Sen DRT9 hexameric ribonucleoprotein complex

SupramoleculeName: Sen DRT9 hexameric ribonucleoprotein complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Salmonella enterica (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: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 58.318961 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MKLEQQIQRV ILEEAKALIK DYHEYHNRVH LESVRNKKRL GDSAPDKKIH RPNYWSFDKK FDPFYVKSNY KSIARSIANK IENRTYLPN EPFTKDVPKP DGGIRKVSIY QIPDAAISKL FFNRLLAKNR HRFSSFSYAY RNDRNVHFAI QDISVDLKKN E RTFLAEFD ...String:
MKLEQQIQRV ILEEAKALIK DYHEYHNRVH LESVRNKKRL GDSAPDKKIH RPNYWSFDKK FDPFYVKSNY KSIARSIANK IENRTYLPN EPFTKDVPKP DGGIRKVSIY QIPDAAISKL FFNRLLAKNR HRFSSFSYAY RNDRNVHFAI QDISVDLKKN E RTFLAEFD FSDFFGSISH SFLNEQFNEN GFYISPEEKF IIRSFLRERK VGIPQGTSIS LFLANLTCWK LDQDLEREGV KF SRYADDT IIWSQEYSKI CNAFNIITNF SKSAGIKINP KKSEGISLLT KKGLPSEITS KNNLDFLGYT LSVENVSIKE KSV KKIKKQ ISYILYRNLI QPLKKTSLAG QTIPANDRDK NFLIAICEIR RYMYGGLSKS QIKDYLSGRS NRLYFKGIMS FYPL VNDVE QLKQLDGWIV SVIYRALKLR CQLLSKWGYN RSHNFPFILD REDIVDKCSK KTIAGRKLFE IPSFLLIHKA LQKGL QESG IEKIMNPQSL NYDYE

UniProtKB: RNA-dependent DNA polymerase

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Macromolecule #2: RNA (141-MER)

MacromoleculeName: RNA (141-MER) / type: rna / ID: 2 / Number of copies: 6
Source (natural)Organism: Salmonella enterica (bacteria)
Molecular weightTheoretical: 52.673969 KDa
SequenceString:
AUUCUCUCAU AGGGAUAACG GUGUGGCCUU CUACCUGUUA GAAAUAAUGG GUCUUCAGUU GUAAUUCGUU GCAACUGACG GGGGGGUGG UGUCAAAGCC GUUUCAACCA AGUGGUAACU UACUUUUACU UGGGUUUAUA CCGUGGAAAA GCCUGAGUCU A ACUC

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
SoftwareName: SerialEM
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 59.8 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6029711
CTF correctionSoftware - Name: cryoSPARC (ver. v4.6.2) / Software - details: Patch-CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.6.2) / Number images used: 500162
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v4.6.2)
Final 3D classificationNumber classes: 3 / Avg.num./class: 382156 / Software - Name: cryoSPARC (ver. v4.6.2)
Details: 3 Class heterogenous refinement used to remove final junk particles
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The initial model for the RT was produced using AlphaFold 3
SoftwareName: UCSF ChimeraX (ver. 1.9)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 101.4 / Target criteria: Cross-correlation coefficient
Output model

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

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