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- PDB-8que: Structure of the Bacteriophage PhiKZ non-virion RNA Polymerase bo... -

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Basic information

Entry
Database: PDB / ID: 8que
TitleStructure of the Bacteriophage PhiKZ non-virion RNA Polymerase bound to DNA and RNA
Components
  • (DNA) x 3
  • DNA-directed RNA polymerase
  • PHIKZ055
  • PHIKZ068
  • PHIKZ074
  • PHIKZ123
  • RNA
KeywordsRNA BINDING PROTEIN / PhiKZ / nvRNAP / RNA / DNA / transcription
Function / homology: / DNA / DNA (> 10) / RNA / PHIKZ123 / PHIKZ074 / PHIKZ068 / PHIKZ055
Function and homology information
Biological speciesPseudomonas phage phiKZ (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
Authorsde 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
DepositionOct 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PHIKZ055
C: DNA-directed RNA polymerase
D: PHIKZ074
E: PHIKZ123
F: DNA
G: DNA
H: RNA
B: PHIKZ068
I: DNA
J: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)391,56112
Polymers391,43010
Non-polymers1312
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area44010 Å2
ΔGint-272 kcal/mol
Surface area98730 Å2
MethodPISA

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Components

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Protein , 5 types, 5 molecules ACDEB

#1: Protein PHIKZ055


Mass: 57976.605 Da / Num. of mol.: 1 / Mutation: N-Terminal-Histidine-Tag
Source method: isolated from a genetically manipulated source
Details: Tetrahedral Zn-CYS binding,Tetrahedral Zn-CYS binding
Source: (gene. exp.) Pseudomonas phage phiKZ (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8SDA7
#2: Protein DNA-directed RNA polymerase


Mass: 78780.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage phiKZ (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: DNA-directed RNA polymerase
#3: Protein PHIKZ074


Mass: 77513.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Tetrahedral Zn-CYS coordination / Source: (gene. exp.) Pseudomonas phage phiKZ (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8SD88
#4: Protein PHIKZ123


Mass: 62959.090 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage phiKZ (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8SD39
#8: Protein PHIKZ068


Mass: 59419.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminus represents docked AlphaFold2 model (CA only); C-terminus built de novo
Source: (gene. exp.) Pseudomonas phage phiKZ (virus) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8SD94

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DNA chain , 3 types, 4 molecules FGIJ

#5: DNA chain DNA


Mass: 23098.877 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas phage phiKZ (virus)
#6: DNA chain DNA


Mass: 23058.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas phage phiKZ (virus) / References: GenBank: 29134936
#9: DNA chain DNA / Coordinate model: P atoms only


Mass: 3016.672 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: DNA - unregistered sequence / Source: (synth.) Pseudomonas phage phiKZ (virus)

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RNA chain / Non-polymers , 2 types, 3 molecules H

#10: Chemical ChemComp-ZN / ZINC ION


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


Mass: 2589.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas phage phiKZ (virus)

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Details

Has ligand of interestN

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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 transcribing promoter P119L
Type: COMPLEX
Details: Transcription from 5 bp RNA primer halted by withholding UTP
Entity ID: #1-#7, #9 / 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.FormulaBuffer-ID
140 mMTris-Cl1
210 mMMgCl21
35 mMDTT1
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 DNA/RNA template at a 1:1 ratio in the presence of 1 mM ATP, CTP and GTP. Reactions were incubated for 30 min at 37 C before use.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K
Details: Settings: -6 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 15000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 1400 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: 51 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3092
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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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: 1198587
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39919 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: CC
Atomic model building
ID 3D fitting-IDSource nameTypeDetailsAccession code
11AlphaFoldin silico model
21Otherin silico modelidealised B-DNA
31PDBexperimental model7OGP
41PDBexperimental model7OGR
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322079
ELECTRON MICROSCOPYf_angle_d0.50830122
ELECTRON MICROSCOPYf_dihedral_angle_d11.9963290
ELECTRON MICROSCOPYf_chiral_restr0.0413326
ELECTRON MICROSCOPYf_plane_restr0.0043697

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