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- PDB-7oro: La Crosse virus polymerase at replication early-elongation stage -

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Basic information

Entry
Database: PDB / ID: 7oro
TitleLa Crosse virus polymerase at replication early-elongation stage
Components
  • La Crosse virus polymerase
  • RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*C)-3')
  • RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*A)-3')
  • RNA (5'-R(P*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
KeywordsVIRAL 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
RNA-directed RNA polymerase, orthobunyavirus / : / Virus, RNA-directed RNA polymerase L, thumb ring domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / : / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesLa Crosse orthobunyavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsArragain, B. / Durieux Trouilleton, Q. / Baudin, F. / Cusack, S. / Schoehn, G. / Malet, H.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0024-02 France
CitationJournal: 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
DepositionJun 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
H: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*C)-3')
S: RNA (5'-R(P*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
T: RNA (5'-R(P*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
P: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*A)-3')
A: La Crosse virus polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)288,9697
Polymers288,8795
Non-polymers902
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12190 Å2
ΔGint-97 kcal/mol
Surface area81530 Å2

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Components

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RNA chain , 3 types, 4 molecules HSTP

#1: RNA chain RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*C)-3')


Mass: 5466.325 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: 5prime vRNA of La Crosse M segment. Mutation of nucleotides G2, U3, A9 and C10 into C2, G3, C9 and G10
Source: (synth.) La Crosse orthobunyavirus
#2: RNA chain RNA (5'-R(P*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')


Mass: 7882.668 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) La Crosse orthobunyavirus
#3: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*UP*GP*UP*A)-3')


Mass: 2896.774 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) La Crosse orthobunyavirus

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Protein , 1 types, 1 molecules A

#4: Protein La Crosse virus polymerase


Mass: 264751.062 Da / Num. of mol.: 1 / Mutation: H34K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) La Crosse orthobunyavirus / Gene: segment L / Cell line (production host): Hi 5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A5HC98, RNA-directed RNA polymerase

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1La Crosse virus polymerase at replication early-elongation stageCOMPLEX#1-#40MULTIPLE SOURCES
2RNACOMPLEX#1-#21RECOMBINANT
3RNACOMPLEX#31NATURAL
4La Crosse virus polymeraseCOMPLEX#41RECOMBINANT
Molecular weightValue: 0.293 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12La Crosse orthobunyavirus2560547
23La Crosse orthobunyavirus2560547
34La Crosse orthobunyavirus2560547
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12synthetic construct (others)32630
24Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HClTris1
2150 mMNaClSodium chloride1
35 mMbeta-mercaptoethanol2-Mercaptoethanol1
4100 uMATPAdenosine triphosphate1
5100 uMGTP1
6100 uMUTP1
75 mMMgCl21
SpecimenConc.: 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 4 degree with (i) 1.9 uM 5prime 1-17 BPm, (ii) 1.9 uM 3prime vRNA 1-25. LACV-LCItag_H34K bound to vRNAs was incubated with 100 ...Details: 1.3 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4 degree with (i) 1.9 uM 5prime 1-17 BPm, (ii) 1.9 uM 3prime vRNA 1-25. LACV-LCItag_H34K bound to vRNAs was incubated with 100 uM ATP/GTP/UTP and 5mM MgCl2 for 4h at 30 degree
Specimen supportDetails: 25 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage 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: 2927
Image scansMovie frames/image: 60 / Used frames/image: 3-50

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4cryoSPARC3CTF correctionPatch CTP estimation
7Coot0.9.2model fitting
9cryoSPARC3initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1532345
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 417757 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 74.97 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6Z8K

6z8k


Pdb chain-ID: A / Pdb chain residue range: 1-2263

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