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- EMDB-17864: Tilapia Lake Virus polymerase in vRNA elongation state (transcrip... -

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Basic information

Entry
Database: EMDB / ID: EMD-17864
TitleTilapia Lake Virus polymerase in vRNA elongation state (transcriptase conformation)
Map data
Sample
  • Complex: Tilapia lake virus polymerase
    • Protein or peptide: Polymerase acidic protein (PA-like)
    • Protein or peptide: Putative PB1
    • Protein or peptide: RNA-dependent RNA polymerase
    • RNA: 5' vRNA end - vRNA loop (40-mer)
    • RNA: Transcription-like product
  • Ligand: ZINC ION
  • Ligand: [(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-1-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-~{N}-[oxidanyl(phosphonooxy)phosphoryl]phosphonamidic acid
  • 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: 2.96 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
DepositionJul 13, 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_17864.png

Downloads & links

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Map

FileDownload / File: emd_17864.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 300 pix.
= 252. Å		0.84 Å/pix.
x 300 pix.
= 252. Å		0.84 Å/pix.
x 300 pix.
= 252. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.047689907 - 0.08116206
Average (Standard dev.)0.000056105488 (±0.0018967771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17864_msk_1.map
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Additional map: #1

Fileemd_17864_additional_1.map
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Half map: #1

Fileemd_17864_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_17864_half_map_2.map
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Sample components

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Entire : Tilapia lake virus polymerase

EntireName: Tilapia lake virus polymerase
Components
  • Complex: Tilapia lake virus polymerase
    • Protein or peptide: Polymerase acidic protein (PA-like)
    • Protein or peptide: Putative PB1
    • Protein or peptide: RNA-dependent RNA polymerase
    • RNA: 5' vRNA end - vRNA loop (40-mer)
    • RNA: Transcription-like product
  • Ligand: ZINC ION
  • Ligand: [(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-1-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-~{N}-[oxidanyl(phosphonooxy)phosphoryl]phosphonamidic acid
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Tilapia lake virus polymerase

SupramoleculeName: Tilapia lake virus polymerase / 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: 5' vRNA end - vRNA loop (40-mer)

MacromoleculeName: 5' vRNA end - vRNA loop (40-mer) / type: rna / ID: 4 / Number of copies: 2
Source (natural)Organism: Tilapia lake virus
Molecular weightTheoretical: 12.736518 KDa
SequenceString:
GCAAAUCUUU CUCACGUCCU GACUUGUGAG 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: 6.675037 KDa
SequenceString:
AGAAUAUAAU ACCAAAUUUU A

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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: [(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-...

MacromoleculeName: [(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-1-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-~{N}-[oxidanyl(phosphonooxy)phosphoryl]phosphonamidic acid
type: ligand / ID: 7 / Number of copies: 1 / Formula: A0I
Molecular weightTheoretical: 482.172 Da
Chemical component information

ChemComp-A0I:
[(2~{R},3~{S},4~{R},5~{R})-5-(4-azanyl-2-oxidanylidene-pyrimidin-1-yl)-3,4-bis(oxidanyl)oxolan-2-yl]methoxy-~{N}-[oxidanyl(phosphonooxy)phosphoryl]phosphonamidic acid

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / 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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 29892
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8psx:
Tilapia Lake Virus polymerase in vRNA elongation state (transcriptase conformation)

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