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- EMDB-18772: Tilapia Lake Virus polymerase in vRNA pre-termination state (tran... -

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Basic information

Entry
Database: EMDB / ID: EMD-18772
TitleTilapia Lake Virus polymerase in vRNA pre-termination state (transcriptase conformation)
Map data
Sample
  • Complex: Tilapia lake virus polymerase heterotrimeric complex
    • Protein or peptide: Polymerase acidic protein (PA-like)
    • Protein or peptide: Putative PB1
    • Protein or peptide: RNA-dependent RNA polymerase
    • RNA: Template vRNA_S loop (40-mer)
    • RNA: Transcription-like product
  • Ligand: ZINC ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsViral polymerase / VIRAL PROTEIN
Function / homologyRNA-directed RNA polymerase activity / DNA/RNA polymerase superfamily / Uncharacterized protein / Putative PB1 / RNA-dependent RNA polymerase
Function and homology information
Biological speciesTilapia lake virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsArragain B / Cusack S
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE11-0028 France
CitationJournal: Nat Commun / Year: 2023
Title: Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family.
Authors: Benoit Arragain / Martin Pelosse / Albert Thompson / Stephen Cusack /
Abstract: Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ...Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated.
History
DepositionOct 26, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18772.png

Downloads & links

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Map

FileDownload / File: emd_18772.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 340 pix.
= 298.52 Å		0.88 Å/pix.
x 340 pix.
= 298.52 Å		0.88 Å/pix.
x 340 pix.
= 298.52 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.878 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.862517 - 3.0919743
Average (Standard dev.)0.002083469 (±0.071596496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 298.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18772_msk_1.map
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Additional map: #1

Fileemd_18772_additional_1.map
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Half map: #2

Fileemd_18772_half_map_1.map
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Half map: #1

Fileemd_18772_half_map_2.map
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Sample components

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Entire : Tilapia lake virus polymerase heterotrimeric complex

EntireName: Tilapia lake virus polymerase heterotrimeric complex
Components
  • Complex: Tilapia lake virus polymerase heterotrimeric complex
    • Protein or peptide: Polymerase acidic protein (PA-like)
    • Protein or peptide: Putative PB1
    • Protein or peptide: RNA-dependent RNA polymerase
    • RNA: Template vRNA_S loop (40-mer)
    • RNA: Transcription-like product
  • Ligand: ZINC ION
  • Ligand: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Tilapia lake virus polymerase heterotrimeric complex

SupramoleculeName: Tilapia lake virus polymerase heterotrimeric complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Tilapia lake virus

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Macromolecule #1: Polymerase acidic protein (PA-like)

MacromoleculeName: Polymerase acidic protein (PA-like) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 47.780508 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDSRFAQLTG VFCDDFTYSE GSRRFLSSYS TVERRPGVPV EGDCYDCLKN KWIAFELEGQ PRKFPKATVR CILNNDATYV CSEQEYQQI CKVQFKDYLE IDGVVKVGHK ASYDAELRER LLELPHPKSG PKPRIEWVAP PRLADISKET AELKRQYGFF E CSKFLACG ...String:
MDSRFAQLTG VFCDDFTYSE GSRRFLSSYS TVERRPGVPV EGDCYDCLKN KWIAFELEGQ PRKFPKATVR CILNNDATYV CSEQEYQQI CKVQFKDYLE IDGVVKVGHK ASYDAELRER LLELPHPKSG PKPRIEWVAP PRLADISKET AELKRQYGFF E CSKFLACG EECGLDQEAR ELILNEYARD REFEFRNGGW IQRYTVASHK PATQKILPLP ASAPLARELL MLIARSTTQA GK VLHSDNT SILAVPVMRD SGKHSKRRPT ASTHHLVVGL SKPGCEHDFE FDGYRAAVHV MHLDPKQSAN IGEQDFVSTR EIY KLDMLE LPPISRKGDL DRASGLETRW DVILLLECLD STRVSQAVAQ HFNRHRLALS VCKDEFRKGY QLASEIRGTI PLSS LYYSL CAVRLRMTVH PFAR

UniProtKB: Uncharacterized protein

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Macromolecule #2: Putative PB1

MacromoleculeName: Putative PB1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 57.179375 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MWAFQEGVCK GNLLSGPTSM KAPDSAARES IDRASEIMTG KSYNAVHTGD LSKLPNQGES PLRIVDSDLY SERSCCWVIE KEGRVVCKS TTLTRGMTSL LNTTKCSSPS ELICKVLTVE SLSEKIGDTS VEELLSHGRY FKCALRDQER GKPKSRAIFL S HPFFRLLS ...String:
MWAFQEGVCK GNLLSGPTSM KAPDSAARES IDRASEIMTG KSYNAVHTGD LSKLPNQGES PLRIVDSDLY SERSCCWVIE KEGRVVCKS TTLTRGMTSL LNTTKCSSPS ELICKVLTVE SLSEKIGDTS VEELLSHGRY FKCALRDQER GKPKSRAIFL S HPFFRLLS SVVETHARSV LSKVSAVYTA TASAEQRAMM AAQVVESRKH VLNGDCTKYN EAIDADTLLK VWDAIGMGSI GV MLAYMVR RKCVLIKDTL VECPGGMLMG MFNATATLAL QGTTDRFLSF SDDFITSFNS PAELREIEDL LFASCHNLSL KKS YISVAS LEINSCTLTR DGDLATGLGC TAGVPFRGPL VTLKQTAAML SGAVDSGVMP FHSAERLFQI KQQECAYRYN NPTY TTRNE DFLPTCLGGK TVISFQSLLT WDCHPFWYQV HPDGPDTIDQ KVLSVLASKT RRRRTRLEAL SDLDPLVPHR LLVSE SDVS KIRAARQAHL KSLGLEQPTN FNYAIYKAVQ PTAGC

UniProtKB: Putative PB1

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Macromolecule #3: RNA-dependent RNA polymerase

MacromoleculeName: RNA-dependent RNA polymerase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 53.782141 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MSQFGKSFKG RTEVTITEYR SHTVKDVHRS LLTADKSLRK SFCFRNALNQ FLDKDLPLLP IRPKLESRVA VKKSKLRSQL SFRPGLTQE EAIDLYNKGY DGDSVSGALQ DRVVNEPVAY SSADNDKFHR GLAALGYTLA DRAFDTCESG FVRAIPTTPC G FICCGPGS ...String:
MSQFGKSFKG RTEVTITEYR SHTVKDVHRS LLTADKSLRK SFCFRNALNQ FLDKDLPLLP IRPKLESRVA VKKSKLRSQL SFRPGLTQE EAIDLYNKGY DGDSVSGALQ DRVVNEPVAY SSADNDKFHR GLAALGYTLA DRAFDTCESG FVRAIPTTPC G FICCGPGS FKDSLGFVIK IGEFWHMYDG FQHFVAVEDA KFLASKSPSF WLAKRLAKRL NLVPKEDPSV AAAECPCKKV WE ASFARAP TALDPFGGRA FCDQGWVYHR DVGYATANHI SQETLFQQAL SVRNLGPQGS ANVSGSIHTA LDRLRAAYSR GTP ASRSIL QGLANLITPV GENFECDLDK RKLNIKALRS PERYITIEGL VVNLDDVVRG FYLDKAKVTV LSRSKWMGYE DLPQ KPPNG TFYCRKRKAM LLISCSPGTY AKKRKVAVQE DRFKDMRVEN FREVAENMDL NQGSGSENLY FQGHHHHHHH HHH

UniProtKB: RNA-dependent RNA polymerase

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Macromolecule #4: Template vRNA_S loop (40-mer)

MacromoleculeName: Template vRNA_S loop (40-mer) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 12.737502 KDa
SequenceString:
GCAAAUCUUU CUCACGUCCU GAUUUGUGAG UAAAAUUUGG

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Macromolecule #5: Transcription-like product

MacromoleculeName: Transcription-like product / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 10.154124 KDa
SequenceString:
AGAAUAUAAU ACCAAAUUUU ACUCACAAAU CA

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER

MacromoleculeName: PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: G2P
Molecular weightTheoretical: 521.208 Da
Chemical component information

ChemComp-G2P:
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GMP-CPP, energy-carrying molecule analogue*YM

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 107403
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8qz8:
Tilapia Lake Virus polymerase in vRNA pre-termination state (transcriptase conformation)

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