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Yorodumi- EMDB-13038: La Crosse virus polymerase at replication late-elongation stage -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13038 | |||||||||
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Title | La Crosse virus polymerase at replication late-elongation stage | |||||||||
Map data | ||||||||||
Sample |
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Keywords | RNA-dependent RNA polymerase / VIRAL PROTEIN | |||||||||
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 | La Crosse orthobunyavirus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
Authors | Arragain B / Durieux Trouilleton Q | |||||||||
Funding support | France, 1 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13038.map.gz | 5.9 MB | EMDB map data format | |
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Header (meta data) | emd-13038-v30.xml emd-13038.xml | 22.8 KB 22.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13038_fsc.xml | 9.3 KB | Display | FSC data file |
Images | emd_13038.png | 132.3 KB | ||
Filedesc metadata | emd-13038.cif.gz | 8.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13038 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13038 | HTTPS FTP |
-Validation report
Summary document | emd_13038_validation.pdf.gz | 396.8 KB | Display | EMDB validaton report |
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Full document | emd_13038_full_validation.pdf.gz | 396.3 KB | Display | |
Data in XML | emd_13038_validation.xml.gz | 11 KB | Display | |
Data in CIF | emd_13038_validation.cif.gz | 14.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13038 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13038 | HTTPS FTP |
-Related structure data
Related structure data | 7oriMC 7orjC 7orkC 7orlC 7ormC 7ornC 7oroC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_13038.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.145 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
+Entire : La Crosse virus polymerase at late elongation stage
+Supramolecule #1: La Crosse virus polymerase at late elongation stage
+Supramolecule #2: RNA
+Supramolecule #3: RNA
+Supramolecule #4: La Crosse virus polymerase
+Macromolecule #1: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*U)-3')
+Macromolecule #2: RNA (5'-R(P*AP*AP*CP*GP*UP*UP*AP*UP*CP*UP*AP*UP*AP*AP*CP*AP*CP*UP...
+Macromolecule #3: RNA (5'-R(P*AP*UP*AP*GP*AP*UP*AP*AP*CP*GP*UP*U)-3')
+Macromolecule #4: La Crosse virus polymerase
+Macromolecule #5: ZINC ION
+Macromolecule #6: MAGNESIUM ION
+Macromolecule #7: PYROPHOSPHATE 2-
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.45 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: 25 mA | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV | ||||||||||||||||||
Details | 1.7 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4degree with (i) 1.9 uM 5prime 1-17 BPm, (ii) 1.9 uM 3prime vRNA 1-30. LACV-LCItag_H34K bound to vRNAs was incubated with 100 uM ATP/GTP/UTP/CTP and 5mM MgCl2 for 4h at 30degree |
-Electron microscopy
Microscope | TFS GLACIOS |
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Temperature | Min: 70.0 K / Max: 70.0 K |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 3-50 / Number grids imaged: 1 / Number real images: 1848 / Average exposure time: 6.6 sec. / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 36000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |