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- EMDB-13038: La Crosse virus polymerase at replication late-elongation stage -

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Basic information

Entry
Database: EMDB / ID: EMD-13038
TitleLa Crosse virus polymerase at replication late-elongation stage
Map data
Sample
  • Complex: La Crosse virus polymerase at late elongation stage
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*U)-3')
      • RNA: RNA (5'-R(P*AP*AP*CP*GP*UP*UP*AP*UP*CP*UP*AP*UP*AP*AP*CP*AP*CP*UP*AP*CP*U)-3')
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*UP*AP*GP*AP*UP*AP*AP*CP*GP*UP*U)-3')
    • Complex: La Crosse virus polymerase
      • Protein or peptide: La Crosse virus polymerase
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: PYROPHOSPHATE 2-
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-directed RNA polymerase L
Similarity search - Component
Biological speciesLa Crosse orthobunyavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsArragain B / Durieux Trouilleton Q / Baudin F / Cusack S / Schoehn G / Malet H
Funding support France, 1 items
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, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateMar 2, 2022-
Current statusMar 2, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ori
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13038.png

Downloads & links

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Map

FileDownload / File: emd_13038.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.145 Å
Density
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.007
Minimum - Maximum-0.011527072 - 0.028252214
Average (Standard dev.)8.871876e-05 (±0.0009878983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 297.69998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1451.1451.145
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z297.700297.700297.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0120.0280.000

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Supplemental data

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Sample components

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Entire : La Crosse virus polymerase at late elongation stage

EntireName: La Crosse virus polymerase at late elongation stage
Components
  • Complex: La Crosse virus polymerase at late elongation stage
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*U)-3')
      • RNA: RNA (5'-R(P*AP*AP*CP*GP*UP*UP*AP*UP*CP*UP*AP*UP*AP*AP*CP*AP*CP*UP*AP*CP*U)-3')
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*UP*AP*GP*AP*UP*AP*AP*CP*GP*UP*U)-3')
    • Complex: La Crosse virus polymerase
      • Protein or peptide: La Crosse virus polymerase
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: PYROPHOSPHATE 2-

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Supramolecule #1: La Crosse virus polymerase at late elongation stage

SupramoleculeName: La Crosse virus polymerase at late elongation stage / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: La Crosse virus polymerase at late elongation stage in complex with the 5prime promoter, the 3prime promoter and template, nucleotides and the product RNA formed
Molecular weightTheoretical: 291 KDa

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Supramolecule #2: RNA

SupramoleculeName: RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: La Crosse orthobunyavirus
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: La Crosse orthobunyavirus

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Supramolecule #4: La Crosse virus polymerase

SupramoleculeName: La Crosse virus polymerase / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: La Crosse orthobunyavirus
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)

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

MacromoleculeName: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*U)-3') / type: rna / ID: 1
Details: mutated 5vRNA of La Crosse virus segment M Mutation of the nucleotides G2, U3, A9 and C10 of the 5 end into C2, G3, C9 and G10
Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 5.466325 KDa
SequenceString:
ACGAGUGUCG UACCAAG

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Macromolecule #2: RNA (5'-R(P*AP*AP*CP*GP*UP*UP*AP*UP*CP*UP*AP*UP*AP*AP*CP*AP*CP*UP...

MacromoleculeName: RNA (5'-R(P*AP*AP*CP*GP*UP*UP*AP*UP*CP*UP*AP*UP*AP*AP*CP*AP*CP*UP*AP*CP*U)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 9.497634 KDa
SequenceString:
AACGUUAUCU AUACUUGGUA GUACACUACU

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

MacromoleculeName: RNA (5'-R(P*AP*UP*AP*GP*AP*UP*AP*AP*CP*GP*UP*U)-3') / type: rna / ID: 3
Details: Product synthetized by La Crosse virus polymerase when incubated with its 5 RNA promoter, RNA template and nucleotides.
Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 9.623761 KDa
SequenceString:
AGUAGUGUAC UACCAAGUAU AGAUAACGUU

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Macromolecule #4: La Crosse virus polymerase

MacromoleculeName: La Crosse virus polymerase / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 264.751062 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDYQEYQQFL ARINTARDAC VAKDIDVDLL MARKDYFGRE LCKSLNIEYR NDVPFIDIIL DIRPEVDPLT IDAPHITPDN YLYINNVLY IIDYKVSVSN ESSVITYDKY YELTRDISDR LSIPIEIVII RIDPVSRDLH INSDRFKELY PTIVVDINFN Q FFDLKQLL ...String:
MDYQEYQQFL ARINTARDAC VAKDIDVDLL MARKDYFGRE LCKSLNIEYR NDVPFIDIIL DIRPEVDPLT IDAPHITPDN YLYINNVLY IIDYKVSVSN ESSVITYDKY YELTRDISDR LSIPIEIVII RIDPVSRDLH INSDRFKELY PTIVVDINFN Q FFDLKQLL YEKFGDDEEF LLKVAHGDFT LTAPWCKTGC PEFWKHPIYK EFKMSMPVPE RRLFEESVKF NAYESERWNT NL VKIREYT KKDYSEHISK SAKNIFLASG FYKQPNKNEI SEGWTLMVER VQDQREISKS LHDQKPSIHF IWGAHNPGNS NNA TFKLIL LSKSLQSIKG ISTYTEAFKS LGKMMDIGDK AIEYEEFCMS LKSKARSSWK QIMNKKLEPK QINNALVLWE QQFM INNDL IDKSEKLKLF KNFCGIGKHK QFKNKMLEDL EVSKPKILDF DDANMYLASL TMMEQSKKIL SKSNGLKPDN FILNE FGSR IKDANKETYD NMHKIFETGY WQCISDFSTL MKNILSVSQY NRHNTFRIAM CANNNVFAIV FPSADIKTKK ATVVYS IIV LHKEEENIFN PGCLHGTFKC MNGYISISRA IRLDKERCQR IVSSPGLFLT TCLLFKHDNP TLVMSDIMNF SIYTSLS IT KSVLSLTEPA RYMIMNSLAI SSNVKDYIAE KFSPYTKTLF SVYMTRLIKN ACFDAYDQRQ RVQLRDIYLS DYDITQKG I KDNRELTSIW FPGSVTLKEY LTQIYLPFYF NAKGLHEKHH VMVDLAKTIL EIECEQRENI KEIWSTNCTK QTVNLKILI HSLCKNLLAD TSRHNHLRNR IENRNNFRRS ITTISTFTSS KSCLKIGDFR KEKELQSVKQ KKILEVQSRK MRLANPMFVT DEQVCLEVG HCNYEMLRNA MPNYTDYIST KVFDRLYELL DKKVLTDKPV IEQIMDMMID HKKFYFTFFN KGQKTSKDRE I FVGEYEAK MCMYAVERIA KERCKLNPDE MISEPGDGKL KVLEQKSEQE IRFLVETTRQ KNREIDEAIE ALATEGSGWS HP QFEKGSG YESNLGKIEK LSLGKAKGLK MEINADMSKW SAQDVFYKYF WLIALDPILY PQEKERILYF MCNYMDKELI LPD ELLFNL LDQKVAYQND IIATMTNQLN SNTVLIKRNW LQGNFNYTSS YVHSCAMSVY KEILKEAITL LDGSILVNSL VHSD DNQTS ITIVQDKMEN DKIIDFAMKE FERACLTFGC QANMKKTYVT NCIKEFVSLF NLYGEPFSIY GRFLLTSVGD CAYIG PYED LASRISSAQT AIKHGCPPSL AWVSIAISHW MTSLTYNMLP GQSNDPIDYF PAENRKDIPI ELNGVLDAPL SMISTV GLE SGNLYFLIKL LSKYTPVMQK RESVVNQIAE VKNWKVEDLT DNEIFRLKIL RYLVLDAEMD PSDIMGETSD MRGRSIL TP RKFTTAGSLR KLYSFSKYQD RLSSPGGMVE LFTYLLEKPE LLVTKGEDMK DYMESVIFRY NSKRFKESLS IQNPAQLF I EQILFSHKPV IDFSGIRDKY INLHDSRALE KEPDILGKVT FTEAYRLLMR DLSSLELTND DIQVIYSYII LNDPMMITI ANTHILSIYG SPQRRMGMSC STMPEFRNLK LIHHSPALVL RAYSKNNPDI QGADPTEMAR DLVHLKEFVE NTNLEEKMKV RIAMNEAEK GQRDIVFELK EMTRFYQVCY EYVKSTEHKI KVFILPAKSY TTTDFCSLMQ GNLIKDKEWY TVHYLKQILS G GHKAIMQH NATSEQNIAF ECFKLITHFA DSFIDSLSRS AFLQLIIDEF SYKDVKVSKL YDIIKNGYNR TDFIPLLFRT GD LRQADLD KYDAMKSHER VTWNDWQTSR HLDMGSINLT ITGYNRSITI IGEDNKLTYA ELCLTRKTPE NITISGRKLL GSR HGLKFE NMSKIQTYPG NYYITYRKKD RHQFVYQIHS HESITRRNEE HMAIRTRIYN EITPVCVVNV AEVDGDQRIL IRSL DYLNN DIFSLSRIKV GLDEFATIKK AHFSKMVSFE GPPIKTGLLD LTELMKSQDL LNLNYDNIRN SNLISFSKLI CCEGS DNIN DGLEFLSDDP MNFTEGEAIH STPIFNIYYS KRGERHMTYR NAIKLLIERE TKIFEEAFTF SENGFISPEN LGCLEA VVS LIKLLKTNEW STVIDKCIHI CLIKNGMDHM YHSFDVPKCF MGNPITRDIN WVMFREFINS LPGTDIPPWN VMTENFK KK CIALINSKFE TQRDFSEFTK LMKKEGGRSN IEFD

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PYROPHOSPHATE 2-

MacromoleculeName: PYROPHOSPHATE 2- / type: ligand / ID: 7 / Number of copies: 1 / Formula: POP
Molecular weightTheoretical: 175.959 Da
Chemical component information

ChemComp-POP:
PYROPHOSPHATE 2- / Pyrophosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.45 mg/mL
BufferpH: 8
Component:
ConcentrationName
50.0 mMTris-HClTris
150.0 mMNaClSodium chloride
5.0 mMbeta-mercaptoethanol2-Mercaptoethanol
100.0 uMATPAdenosine triphosphate
100.0 uMGTP
100.0 uMCTP
100.0 uMUTP
5.0 mMMgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details1.7 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

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm 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

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Image processing

Particle selectionNumber selected: 1200554
CTF correctionSoftware - Name: cryoSPARC (ver. 3.0) / Software - details: Patch CTP estimation
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 24151
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6z8k


Chain - Chain ID: A / Chain - Residue range: 1-2263
RefinementProtocol: AB INITIO MODEL / Overall B value: 109.75
Output model

PDB-7ori:
La Crosse virus polymerase at replication late-elongation stage

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