[English] 日本語
Yorodumi- PDB-7orj: La Crosse virus polymerase at transcription capped RNA cleavage stage -
+Open data
-Basic information
Entry | Database: PDB / ID: 7orj | ||||||
---|---|---|---|---|---|---|---|
Title | La Crosse virus polymerase at transcription capped RNA cleavage stage | ||||||
Components |
| ||||||
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 orthobunyavirus | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | ||||||
Authors | Arragain, B. / Durieux Trouilleton, Q. / Baudin, F. / Cusack, S. / Schoehn, G. / Malet, H. | ||||||
Funding support | France, 1items
| ||||||
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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7orj.cif.gz | 419.4 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb7orj.ent.gz | 328.2 KB | Display | PDB format |
PDBx/mmJSON format | 7orj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7orj_validation.pdf.gz | 880.5 KB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 7orj_full_validation.pdf.gz | 906.2 KB | Display | |
Data in XML | 7orj_validation.xml.gz | 64.2 KB | Display | |
Data in CIF | 7orj_validation.cif.gz | 97.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/or/7orj ftp://data.pdbj.org/pub/pdb/validation_reports/or/7orj | HTTPS FTP |
-Related structure data
Related structure data | 13039MC 7oriC 7orkC 7orlC 7ormC 7ornC 7oroC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data | |
EM raw data | EMPIAR-10995 (Title: Cryo-EM data used for the determination of LACV-L in transcription capped primer cleavage state Data size: 934.6 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 |
|
---|---|
1 |
|
-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 |
---|
-RNA chain , 3 types, 3 molecules HTP
#2: RNA chain | Mass: 5466.325 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: Mutated 5prime vRNA of La Crosse virus M segment. nNucleotides G2, U3, A9 and C10 are mutated into C2, G3, C9 and G10 Source: (synth.) La Crosse orthobunyavirus |
---|---|
#3: RNA chain | Mass: 7882.668 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus |
#4: RNA chain | Mass: 4935.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus |
-Non-polymers , 2 types, 2 molecules
#5: Chemical | ChemComp-ZN / |
---|---|
#6: Chemical | ChemComp-ATP / |
-Details
Has ligand of interest | Y |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: La Crosse virus polymerase at transcription capped RNA cleavage stage Type: COMPLEX Details: La Crosse virus polymerase aat transcription cleavage stage in complex with the 5prime promoter, the 3prime promoter and template and the capped RNA bound directed towards the endonuclease for cleavage Entity ID: #1-#4 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 0.284 MDa / Experimental value: NO | ||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||
Buffer component |
| ||||||||||||||||||||||||||||
Specimen | Conc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 1.3 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4degree 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-LCItag_ ...Details: 1.3 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4degree 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-LCItag_H34K bound to vRNAs and capped primer was incubated with 100 uM ATP/UTP and 2mM MgCl2 for 1h at 30degree. | ||||||||||||||||||||||||||||
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 |
-Electron microscopy imaging
Microscopy | Model: TFS GLACIOS |
---|---|
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 36000 X / Calibrated magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 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 | Average exposure time: 6.6 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3270 |
Image scans | Movie frames/image: 60 / Used frames/image: 3-50 |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1852232 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29065 / Algorithm: BACK PROJECTION / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 60.25 / Protocol: AB INITIO MODEL / Space: REAL | ||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6Z6G Pdb chain-ID: A / Accession code: 6Z6G / Pdb chain residue range: 1-2263 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
|