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- PDB-9pnw: N4 vRNAP gp50 bound to P1 promoter - closed complex -

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Basic information

Entry
Database: PDB / ID: 9pnw
TitleN4 vRNAP gp50 bound to P1 promoter - closed complex
Components
  • N4 P1 DNA Promoter
  • Virion DNA-directed RNA polymerase
KeywordsVIRAL PROTEIN / TRANSFERASE / single-subunit RNAP polymerase / bacteriophage ejection protein
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / virion component / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / GTP binding / ATP binding / metal ion binding
Similarity search - Function
: / : / : / : / Virion DNA-directed RNA polymerase, plug insertion / Virion DNA-directed RNA polymerase domain / Virion DNA-directed RNA polymerase domain / Bacteriophage N4 RNA polymerase, helical domain
Similarity search - Domain/homology
DNA / DNA (> 10) / Virion DNA-directed RNA polymerase
Similarity search - Component
Biological speciesEscherichia phage N4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBellis, N.F. / Lokareddy, R.K. / Cingolani, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140733 United States
National Institutes of Health/Office of the DirectorOD024978 United States
CitationJournal: Res Sq / Year: 2025
Title: Structure of the giant RNA polymerase ejected from coliphage N4.
Authors: Nathan F Bellis / Ravi K Lokareddy / Mikhail Pavlenok / Stephanie L Cooper Horton / James L Kizziah / Francesca Forti / David A Schneider / Michael Niederweis / Federica Briani / Gino Cingolani /
Abstract: are widespread prokaryotic viruses that encapsidate a giant (~3,500-residue) virion-associated RNA polymerase (vRNAP). During infection, vRNAP is expelled into Gram-negative bacteria, along with two ... are widespread prokaryotic viruses that encapsidate a giant (~3,500-residue) virion-associated RNA polymerase (vRNAP). During infection, vRNAP is expelled into Gram-negative bacteria, along with two additional ejection proteins, to assemble a transient DNA-ejectosome that becomes transcriptionally active, initiating viral replication. Here, we present an integrative structural analysis of the coliphage N4 vRNAP (gp50). We find that this 383 kDa enzyme is a multi-domain, single-chain RNA polymerase, structurally distinct from both compact single-chain RNAPs and large multi-subunit holoenzymes. vRNAP is composed of loosely connected domains and exhibits an intramolecular mode of allosteric regulation through its C-terminal domain. Comparative analysis of intact and genome-released virions identified gp51, which forms an outer-membrane complex, and gp52, which assembles a periplasmic tunnel. These proteins generate heterogeneous pores that facilitate the release of vRNAP. We further uncover a signaling hub in the phage tail, composed of the receptor-binding protein, tail tube, and tail plug, that detects receptor engagement and orchestrates the release of ejection proteins. We propose that the beads-on-a-string architecture of vRNAP enables the translocation of megadalton-scale protein complexes through the ~35 Å channel formed by the tail and ejection proteins. These findings establish N4 as a distinctive model for protein translocation through biological channels.
History
DepositionJul 21, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: N4 P1 DNA Promoter
A: Virion DNA-directed RNA polymerase


Theoretical massNumber of molelcules
Total (without water)393,3412
Polymers393,3412
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain N4 P1 DNA Promoter


Mass: 10437.731 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage N4 (virus)
#2: Protein Virion DNA-directed RNA polymerase / vRNAP / Gene product 50 / gp50


Mass: 382903.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage N4 (virus) / Gene: 50 / Production host: Escherichia coli (E. coli) / References: UniProt: Q859P9, DNA-directed RNA polymerase
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: N4 virion-associated RNA polymerase gp50 closed complex bound to P1 ssDNA hairpin promoter
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia phage N4 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21_5207:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128569 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219743
ELECTRON MICROSCOPYf_angle_d0.4226765
ELECTRON MICROSCOPYf_dihedral_angle_d9.3722859
ELECTRON MICROSCOPYf_chiral_restr0.0353049
ELECTRON MICROSCOPYf_plane_restr0.0033419

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