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- PDB-7ork: La Crosse virus polymerase in transcription mode with cleaved cap... -

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Basic information

Entry
Database: PDB / ID: 7ork
TitleLa Crosse virus polymerase in transcription mode with cleaved capped RNA entering the polymerase active site
Components
  • RNA (5'-D(*(GTG))-R(P*A)-3')
  • RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
  • RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
  • RNA-directed RNA polymerase L
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
ADENOSINE-5'-TRIPHOSPHATE / RNA / RNA (> 10) / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesBunyavirus La Crosse
La Crosse virus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
H: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
T: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
P: RNA (5'-D(*(GTG))-R(P*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)283,6578
Polymers283,0364
Non-polymers6214
Water86548
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11280 Å2
ΔGint-87 kcal/mol
Surface area89190 Å2
MethodPISA

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Components

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Protein , 1 types, 1 molecules A

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


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 RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')


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


Mass: 7882.668 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) La Crosse virus
#4: RNA chain RNA (5'-D(*(GTG))-R(P*A)-3')


Mass: 4935.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 4 types, 52 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 48 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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

ComponentName: 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
Molecular weightValue: 0.284 MDa / Experimental value: NO
Buffer solutionpH: 8
Details: 50 mM TRIS-HCl pH 8, 150 mM NaCl, 5 mM BME, 100 uM ATP/UTP and 5mM MgCl2.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-Hcl1
2150 mMNaClNaCl1
35 mMbeta-mercaptoethanol1
4100 uMATP1
5100 uMUTP1
65 mMMgCl21
SpecimenConc.: 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 supportDetails: 25mA / 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 / Details: blot force 1 2 sec

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron 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 opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4cryoSPARC3CTF correctionPatch CTF correction
7Coot0.9.2model fitting
9PHENIX1.19.2model refinementreal space refinement
10cryoSPARC3initial Euler assignment
11RELION3.1final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4615689
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 76579 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 60.74 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6Z6G

6z6g


Pdb chain-ID: A / Accession code: 6Z6G / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00419109
ELECTRON MICROSCOPYf_angle_d0.50225966
ELECTRON MICROSCOPYf_dihedral_angle_d7.8312864
ELECTRON MICROSCOPYf_chiral_restr0.0412919
ELECTRON MICROSCOPYf_plane_restr0.0033146

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