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- EMDB-13043: La Crosse virus polymerase at replication initiation stage -

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Basic information

Entry
Database: EMDB / ID: EMD-13043
TitleLa Crosse virus polymerase at replication initiation stage
Map data
Sample
  • Complex: La Crosse virus polymerase at replication initiation
    • Complex: La Crosse virus polymerase
      • Protein or peptide: La Crosse virus polymerase
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
      • RNA: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsRNA-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
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: 2.8 Å
AuthorsArragain B / Durieux Trouilleton Q
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-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7orn
  • Surface level: 0.0045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13043.png

Downloads & links

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Map

FileDownload / File: emd_13043.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 440 pix.
= 283.8 Å		0.65 Å/pix.
x 440 pix.
= 283.8 Å		0.65 Å/pix.
x 440 pix.
= 283.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.645 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0045 / Movie #1: 0.0045
Minimum - Maximum-0.022140741 - 0.046043396
Average (Standard dev.)0.00003396581 (±0.0008216498)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 283.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6450.6450.645
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z283.800283.800283.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.0220.0460.000

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Supplemental data

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Sample components

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Entire : La Crosse virus polymerase at replication initiation

EntireName: La Crosse virus polymerase at replication initiation
Components
  • Complex: La Crosse virus polymerase at replication initiation
    • Complex: La Crosse virus polymerase
      • Protein or peptide: La Crosse virus polymerase
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
      • RNA: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: La Crosse virus polymerase at replication initiation

SupramoleculeName: La Crosse virus polymerase at replication initiation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 279 KDa

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Supramolecule #2: La Crosse virus polymerase

SupramoleculeName: La Crosse virus polymerase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: La Crosse orthobunyavirus

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: La Crosse orthobunyavirus

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Macromolecule #1: La Crosse virus polymerase

MacromoleculeName: La Crosse virus polymerase / type: protein_or_peptide / ID: 1 / 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

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*CP*AP*AP*G)-3')
type: rna / ID: 2
Details: Mutated 5' sequence of La Crosse virus M segment Nucleotides G2, U3, A9 and C10 were mutated 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 #3: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*UP*UP*GP*GP*UP*AP*GP*UP*AP*CP*AP*CP*UP*AP*CP*U)-3')
type: rna / ID: 3
Details: 3' extremity (25 nucleotides) of La Crosse virus M segment
Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 7.882668 KDa
SequenceString:
UAUCUAUACU UGGUAGUACA CUACU

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 92 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.35 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris-Hcl
150.0 mMNaClNaCl
5.0 mMbeta-mercaptoethanol
100.0 uMATP
100.0 uMUTP
5.0 mMMgCl2

Details: 50 mM TRIS-HCl pH 8, 150 mM NaCl, 5 mM BME, 100 uM ATP/UTP and 5mM MgCl2.
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 1 2 sec.
Details1.3 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. 100 uM ATP/UTP and 5mM MgCl2 for 4h at 30dregree

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 70.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 15573 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 18.0 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 18.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4615689
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 641008
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6z6g


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 64.15
Output model

PDB-7orn:
La Crosse virus polymerase at replication initiation stage

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