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Yorodumi- PDB-7ork: La Crosse virus polymerase in transcription mode with cleaved cap... -
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-Basic information
Entry | Database: PDB / ID: 7ork | ||||||
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Title | La Crosse virus polymerase in transcription mode with cleaved capped RNA entering the polymerase active site | ||||||
Components |
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Keywords | VIRAL PROTEIN / RNA-dependent RNA polymerase | ||||||
Function / homology | Function and homology information host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding ...host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding Similarity search - Function | ||||||
Biological species | Bunyavirus La Crosse La Crosse virus Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Arragain, B. / Durieux Trouilleton, Q. / Baudin, F. / Cusack, S. / Schoehn, G. / Malet, H. | ||||||
Funding support | France, 1items
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Citation | Journal: Nat Commun / Year: 2022 Title: Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription. Authors: Benoît Arragain / Quentin Durieux Trouilleton / Florence Baudin / Jan Provaznik / Nayara Azevedo / Stephen Cusack / Guy Schoehn / Hélène Malet / Abstract: Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse ...Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ork.cif.gz | 430.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ork.ent.gz | 337.3 KB | Display | PDB format |
PDBx/mmJSON format | 7ork.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ork_validation.pdf.gz | 869.9 KB | Display | wwPDB validaton report |
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Full document | 7ork_full_validation.pdf.gz | 885.2 KB | Display | |
Data in XML | 7ork_validation.xml.gz | 66 KB | Display | |
Data in CIF | 7ork_validation.cif.gz | 102.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/or/7ork ftp://data.pdbj.org/pub/pdb/validation_reports/or/7ork | HTTPS FTP |
-Related structure data
Related structure data | 13040MC 7oriC 7orjC 7orlC 7ormC 7ornC 7oroC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | |
EM raw data | EMPIAR-11021 (Title: Cryo-EM data used for the determination of the structures of LACV-L in 3 different states: replication initiation state, transcription capped primer active site entry state and transcription initiation state Data size: 2.2 TB Data #1: Unaligned multiframe micrographs of LACV-L incubated with capped RNA 14-mer, 3' vRNA 1-25, 5' 1-17BPm, UTP, ATP, MgCl2 [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 1 types, 1 molecules A
#1: Protein | Mass: 264751.062 Da / Num. of mol.: 1 / Mutation: H34K Source method: isolated from a genetically manipulated source Source: (gene. exp.) Bunyavirus La Crosse / Cell line (production host): Hi 5 / Production host: Trichoplusia ni (cabbage looper) References: UniProt: A5HC98, RNA-directed RNA polymerase, Hydrolases; Acting on ester bonds |
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-RNA chain , 3 types, 3 molecules HTP
#2: RNA chain | Mass: 5466.325 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: 5prime vRNA extremity of La Crosse virus M segment. Nucleotides G2, U3, A9 and C10 mutated into C2, G3, C9 and G10 Source: (synth.) La Crosse virus |
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#3: RNA chain | Mass: 7882.668 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) La Crosse virus |
#4: RNA chain | Mass: 4935.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
-Non-polymers , 4 types, 52 molecules
#5: Chemical | #6: Chemical | ChemComp-ZN / | #7: Chemical | ChemComp-ATP / | #8: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | Y |
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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: La Crosse virus polymerase in transcription mode with cleaved capped RNA entering the polymerase active site Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.284 MDa / Experimental value: NO | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 8 Details: 50 mM TRIS-HCl pH 8, 150 mM NaCl, 5 mM BME, 100 uM ATP/UTP and 5mM MgCl2. | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 1.3 uM LACV-L FLCI-Strep were sequentially incubated for 1h at 4 degree with (i) 1.9 uM 5prime 1-17 BPm and 3.9 uM commercial 14-mer capped primer, (ii) 1.9 uM 3prime vRNA 1-25. LACV-L FLCI- ...Details: 1.3 uM LACV-L FLCI-Strep were sequentially incubated for 1h at 4 degree with (i) 1.9 uM 5prime 1-17 BPm and 3.9 uM commercial 14-mer capped primer, (ii) 1.9 uM 3prime vRNA 1-25. LACV-L FLCI-Strep bound to vRNAs and capped primer was incubated with 100 uM ATP/UTP and 2mM MgCl2 for 1h at 30 degree. | |||||||||||||||||||||||||||||||||||
Specimen support | Details: 25mA / 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: 100 % / Chamber temperature: 293 K / Details: blot force 1 2 sec |
-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: 130000 X / Calibrated magnification: 130000 X / Nominal defocus max: 18000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 18000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15573 |
EM imaging optics | Energyfilter name: GIF Bioquantum |
Image scans | Width: 5760 / Height: 4092 |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4615689 | |||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76579 / Algorithm: BACK PROJECTION / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 60.74 / Protocol: AB INITIO MODEL / Space: REAL | |||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6Z6G Pdb chain-ID: A / Accession code: 6Z6G / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||||||||||||||||||||
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