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- PDB-9rjs: Structure of the Bacteriophage PhiKZ non-virion RNA Polymerase bo... -

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Basic information

Entry
Database: PDB / ID: 9rjs
TitleStructure of the Bacteriophage PhiKZ non-virion RNA Polymerase bound to an analogue of its promoter
Components
  • DNA - ATGAGTAATTTTAGTGAATGTATTTGCTATATTGCTATGTAGACAGTTCCCAAAAGCCTAAAGTTACAATATAGG
  • DNA - CCTATATTGTAACTTTAGGCTTTTGGGAACTCCTCTCATATTCCCATAGCAAATACATTCACTAAAATTACTCAT
  • DNA-directed RNA polymerase,PHIKZ056.1
  • PHIKZ068
  • PHIKZ071
  • PHIKZ074
  • PHIKZ123
KeywordsRNA BINDING PROTEIN / PhiKZ / nvRNAP / RNA / DNA / transcription
Function / homologyDNA / DNA (> 10) / DNA-directed RNA polymerase / PHIKZ056.1 / PHIKZ123 / PHIKZ074 / PHIKZ068
Function and homology information
Biological speciesPhikzvirus phiKZ
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsChen, C. / de Martin Garrido, N. / Yakunina, M. / Aylett, C.H.S.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust206212/Z/17/A United Kingdom
Royal Society206212/Z/17/A United Kingdom
CitationJournal: J Mol Biol / Year: 2024
Title: Structure of the Bacteriophage PhiKZ Non-virion RNA Polymerase Transcribing from its Promoter p119L.
Authors: Natàlia de Martín Garrido / Chao-Sheng Chen / Kailash Ramlaul / Christopher H S Aylett / Maria Yakunina /
Abstract: Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people ...Bacteriophage ΦKZ (PhiKZ) is the founding member of a family of giant bacterial viruses. It has potential as a therapeutic as its host, Pseudomonas aeruginosa, kills tens of thousands of people worldwide each year. ΦKZ infection is independent of the host transcriptional apparatus; the virus forms a "nucleus", producing a proteinaceous barrier around the ΦKZ genome that excludes the host immune systems. It expresses its own non-canonical multi-subunit non-virion RNA polymerase (nvRNAP), which is imported into its "nucleus" to transcribe viral genes. The ΦKZ nvRNAP is formed by four polypeptides representing homologues of the eubacterial β/β' subunits, and a fifth that is likely to have evolved from an ancestral homologue to σ-factor. We have resolved the structure of the ΦKZ nvRNAP initiating transcription from its cognate promoter, p119L, including previously disordered regions. Our results shed light on the similarities and differences between ΦKZ nvRNAP mechanisms of transcription and those of canonical eubacterial RNAPs and the related non-canonical nvRNAP of bacteriophage AR9.
History
DepositionJun 12, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase,PHIKZ056.1
B: PHIKZ068
C: PHIKZ071
D: PHIKZ074
E: PHIKZ123
X: DNA - ATGAGTAATTTTAGTGAATGTATTTGCTATATTGCTATGTAGACAGTTCCCAAAAGCCTAAAGTTACAATATAGG
Y: DNA - CCTATATTGTAACTTTAGGCTTTTGGGAACTCCTCTCATATTCCCATAGCAAATACATTCACTAAAATTACTCAT
hetero molecules


Theoretical massNumber of molelcules
Total (without water)382,9049
Polymers382,7737
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 5 types, 5 molecules ABCDE

#1: Protein DNA-directed RNA polymerase,PHIKZ056.1


Mass: 57976.605 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phikzvirus phiKZ / Production host: Escherichia coli (E. coli) / References: UniProt: I7DB47, UniProt: L7T138
#2: Protein PHIKZ068


Mass: 59442.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Numbering correct in original mmCIF file / Source: (gene. exp.) Phikzvirus phiKZ / Production host: Escherichia coli (E. coli) / References: UniProt: Q8SD94
#3: Protein PHIKZ071


Mass: 78780.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phikzvirus phiKZ / Production host: Escherichia coli (E. coli)
#4: Protein PHIKZ074


Mass: 77513.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phikzvirus phiKZ / Production host: Escherichia coli (E. coli) / References: UniProt: Q8SD88
#5: Protein PHIKZ123


Mass: 62959.090 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phikzvirus phiKZ / Production host: Escherichia coli (E. coli) / References: UniProt: Q8SD39

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DNA chain , 2 types, 2 molecules XY

#6: DNA chain DNA - ATGAGTAATTTTAGTGAATGTATTTGCTATATTGCTATGTAGACAGTTCCCAAAAGCCTAAAGTTACAATATAGG


Mass: 23218.951 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Phikzvirus phiKZ
#7: DNA chain DNA - CCTATATTGTAACTTTAGGCTTTTGGGAACTCCTCTCATATTCCCATAGCAAATACATTCACTAAAATTACTCAT


Mass: 22881.719 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Phikzvirus phiKZ

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Non-polymers , 1 types, 2 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
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: PhiKZ non-virion RNA polymerase bound to an analogue of promoter p119L
Type: COMPLEX
Details: Mismatches introduced around the transcription start site to stably open the bubble
Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas phage phiKZ (virus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pET vectors
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
140 mMTris-ClTris-Cl1
210 mMMgCl2MgCl21
35 mMDTTDTT1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: PhiKZ nvRNAP complexes were mixed at 0.1 mg/mL final concentration with the P119L promoter analogue at a 1:1 ratio. Reactions were incubated for 30 min at 25 C before use.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K
Details: Settings: -4 blot force, 4 s waiting time and 0.5-1 s blotting time.

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 20670 / Details: Images collected as TIFF movies
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1EMANparticle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fittingRigid body
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIXmodel refinementReal space refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4178816
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329400 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 104.82 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CC
Atomic model building

3D fitting-ID: 1

IDPDB-IDChain-IDChain residue rangeSource nameTypeDetailsAccession codeInitial refinement model-ID
1B0-278AlphaFoldin silico model
2XOtherin silico modelIdealised B-DNA
3YOtherin silico modelIdealised B-DNA
47OGP

7ogp

PDBexperimental model7OGP3
57OGR

7ogr

PDBexperimental model7OGR4
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.01 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002921696
ELECTRON MICROSCOPYf_angle_d0.410229578
ELECTRON MICROSCOPYf_chiral_restr0.0393256
ELECTRON MICROSCOPYf_plane_restr0.00333621
ELECTRON MICROSCOPYf_dihedral_angle_d14.10783222

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