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Yorodumi- PDB-8que: Structure of the Bacteriophage PhiKZ non-virion RNA Polymerase bo... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8que | |||||||||
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Title | Structure of the Bacteriophage PhiKZ non-virion RNA Polymerase bound to DNA and RNA | |||||||||
Components |
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Keywords | RNA BINDING PROTEIN / PhiKZ / nvRNAP / RNA / DNA / transcription | |||||||||
Function / homology | : / DNA / DNA (> 10) / RNA / PHIKZ123 / PHIKZ074 / PHIKZ068 / PHIKZ055 Function and homology information | |||||||||
Biological species | Pseudomonas phage phiKZ (virus) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | de Martin Garrido, N. / Yakunina, M. / Aylett, C.H.S. | |||||||||
Funding support | United Kingdom, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8que.cif.gz | 521.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8que.ent.gz | 396.7 KB | Display | PDB format |
PDBx/mmJSON format | 8que.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8que_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8que_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8que_validation.xml.gz | 84.9 KB | Display | |
Data in CIF | 8que_validation.cif.gz | 128.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qu/8que ftp://data.pdbj.org/pub/pdb/validation_reports/qu/8que | HTTPS FTP |
-Related structure data
Related structure data | 18661MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 5 types, 5 molecules ACDEB
#1: Protein | 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 |
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#2: Protein | 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 | 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 | 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 | 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 |
-DNA chain , 3 types, 4 molecules FGIJ
#5: DNA chain | Mass: 23098.877 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas phage phiKZ (virus) |
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#6: DNA chain | Mass: 23058.852 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas phage phiKZ (virus) / References: GenBank: 29134936 |
#9: DNA chain | Mass: 3016.672 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: DNA - unregistered sequence / Source: (synth.) Pseudomonas phage phiKZ (virus) |
-RNA chain / Non-polymers , 2 types, 3 molecules H
#10: Chemical | #7: RNA chain | | 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 interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: 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 | ||||||||||||||||
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Molecular weight | Value: 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 solution | pH: 8 | ||||||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||
Vitrification | Instrument: 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron 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 optics | Energyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV |
Image scans | Sampling size: 14 µm / Width: 4096 / Height: 4096 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1198587 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CC | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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