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Yorodumi- PDB-7oqy: Cryo-EM structure of the cellular negative regulator TFS4 bound t... -
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-Basic information
Entry | Database: PDB / ID: 7oqy | ||||||
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Title | Cryo-EM structure of the cellular negative regulator TFS4 bound to the archaeal RNA polymerase | ||||||
Components |
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Keywords | TRANSCRIPTION / negative regulator / inhibitor / Archaea / Sso TFS4 / trancript cleavage factor | ||||||
Function / homology | Function and homology information 3 iron, 4 sulfur cluster binding / RNA polymerase III activity / transcription initiation at RNA polymerase III promoter / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...3 iron, 4 sulfur cluster binding / RNA polymerase III activity / transcription initiation at RNA polymerase III promoter / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / defense response to virus / protein dimerization activity / negative regulation of DNA-templated transcription / nucleotide binding / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Sulfolobus acidocaldarius DSM 639 (acidophilic) Saccharolobus solfataricus (archaea) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.61 Å | ||||||
Authors | Pilotto, S. / Fouqueau, T. / Lukoyanova, N. / Sheppard, C. / Lucas-Staat, S. / Diaz-Santin, L.M. / Matelska, D. / Prangishvili, D. / Cheung, A.C.M. / Werner, F. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: Nat Commun / Year: 2021 Title: Structural basis of RNA polymerase inhibition by viral and host factors. Authors: Simona Pilotto / Thomas Fouqueau / Natalya Lukoyanova / Carol Sheppard / Soizick Lucas-Staat / Luis Miguel Díaz-Santín / Dorota Matelska / David Prangishvili / Alan C M Cheung / Finn Werner / Abstract: RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution ...RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 7oqy.cif.gz | 629.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7oqy.ent.gz | 507.2 KB | Display | PDB format |
PDBx/mmJSON format | 7oqy.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7oqy_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7oqy_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 7oqy_validation.xml.gz | 97.6 KB | Display | |
Data in CIF | 7oqy_validation.cif.gz | 151.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/oq/7oqy ftp://data.pdbj.org/pub/pdb/validation_reports/oq/7oqy | HTTPS FTP |
-Related structure data
Related structure data | 13034MC 7ok0C 7oq4C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA-directed RNA polymerase subunit ... , 10 types, 10 molecules ABCDEHKLNP
#1: Protein | Mass: 99929.055 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOA' (RPO1N) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P11512, DNA-directed RNA polymerase |
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#2: Protein | Mass: 126647.102 Da / Num. of mol.: 1 / Mutation: V772I natural variant / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOB (RPO2) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus Strain: ATCC 33909 / DSM 639 / JCM 8929 / NBRC 15157 / NCIMB 11770 References: UniProt: P11513, DNA-directed RNA polymerase |
#3: Protein | Mass: 44507.430 Da / Num. of mol.: 1 / Source method: isolated from a natural source Details: DNA-dependent RNA polymerase subunit RPOA'' (RPO1C) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P11514, DNA-directed RNA polymerase |
#4: Protein | Mass: 29858.752 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOD (RPO3) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P39471, DNA-directed RNA polymerase |
#5: Protein | Mass: 20496.750 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOE (RPO7) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P39466, DNA-directed RNA polymerase |
#8: Protein | Mass: 9477.059 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOH (RPO5) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P11521, DNA-directed RNA polymerase |
#9: Protein | Mass: 10251.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOK (RPO6) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P39463, DNA-directed RNA polymerase |
#10: Protein | Mass: 10061.791 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOL (RPO11) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P46217, DNA-directed RNA polymerase |
#11: Protein | Mass: 7673.108 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPON (RPO10) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: P39472, DNA-directed RNA polymerase |
#12: Protein/peptide | Mass: 5662.912 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOP (RPO12) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus Strain: ATCC 33909 / DSM 639 / JCM 8929 / NBRC 15157 / NCIMB 11770 References: UniProt: Q4JAE8, DNA-directed RNA polymerase |
-DNA-directed RNA polymerase, subunit ... , 3 types, 3 molecules FGY
#6: Protein | Mass: 13070.987 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPOF (RPO4) Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: Q4JB12, DNA-directed RNA polymerase |
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#7: Protein | Mass: 15292.758 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: DNA-dependent RNA polymerase subunit RPOG (RPO8) Source: (gene. exp.) Sulfolobus acidocaldarius DSM 639 (acidophilic) Gene: rpoG, Saci_0661 Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus Production host: Sulfolobus acidocaldarius DSM 639 (acidophilic) References: UniProt: Q4JAY4, DNA-directed RNA polymerase |
#14: Protein | Mass: 10422.411 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: DNA-directed RNA polymerase subunit RpoM-2 (TFS4) Source: (gene. exp.) Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea) Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: rpoM-2, SSO9221 / Plasmid: pET21a+ Details (production host): Cloned within NdeI and XhoI restriction sites with a stop codon at the end of the protein sequence Production host: Escherichia coli (E. coli) / Strain (production host): DE3 Rosetta2 / References: UniProt: Q97X43, DNA-directed RNA polymerase |
-Protein , 1 types, 1 molecules Q
#13: Protein | Mass: 12331.662 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: DNA-dependent RNA polymerase subunit RPO13 Source: (natural) Sulfolobus acidocaldarius DSM 639 (acidophilic) Plasmid details: genetically manipulated to insert an additional copy of Rpo8 with a hexa-his tag at the C-terminus References: UniProt: Q4JAJ6 |
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-Non-polymers , 3 types, 10 molecules
#15: Chemical | ChemComp-MG / | ||
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#16: Chemical | ChemComp-ZN / #17: Chemical | ChemComp-F3S / | |
-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: TFS4 bound to the RNA polymerase / Type: COMPLEX / Entity ID: #1-#14 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.414 MDa / Experimental value: YES | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Sulfolobus acidocaldarius DSM 639 (acidophilic) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: the sample was applied twice on the grid before vetrification | ||||||||||||||||||||||||||||||
Specimen support | Details: the grid was coated with graphene oxide after to be glow discharged and before applying the sample Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 94 % / Chamber temperature: 277 K |
-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: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2 sec. / Electron dose: 45.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1760 Details: images were collected as movie-stacks of 40 frames in super resolution mode |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: All images were binned 2x, aligned and summed by using MotionCor2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1161535 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 350682 / Num. of class averages: 2 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 7OK0 Accession code: 7OK0 / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
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