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- EMDB-17792: Influenza A/H7N9 polymerase symmetric dimer bound to the promoter... -

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Basic information

Entry
Database: EMDB / ID: EMD-17792
TitleInfluenza A/H7N9 polymerase symmetric dimer bound to the promoter (PA K289A/C489R)
Map data
Sample
  • Complex: Influenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
    • Complex: Polymerase acidic protein, RNA-directed RNA polymerase catalytic subunit and Polymerase basic protein 2
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: 51-mer vRNA loop (v51_mut_S)
      • RNA: 51-mer vRNA loop (v51_mut_S)
  • Ligand: MAGNESIUM ION
KeywordsViral polymerase / VIRAL PROTEIN
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / hydrolase activity ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / hydrolase activity / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain ...Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Polymerase acidic protein / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 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: 2024
Title: The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome.
Authors: Tim Krischuns / Benoît Arragain / Catherine Isel / Sylvain Paisant / Matthias Budt / Thorsten Wolff / Stephen Cusack / Nadia Naffakh /
Abstract: The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation ...The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.
History
DepositionJul 4, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17792.png

Downloads & links

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Map

FileDownload / File: emd_17792.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.)
0.91 Å/pix.
x 340 pix.
= 308.38 Å		0.91 Å/pix.
x 340 pix.
= 308.38 Å		0.91 Å/pix.
x 340 pix.
= 308.38 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.907 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-2.6121275 - 4.0942016
Average (Standard dev.)0.0021651485 (±0.08032841)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 308.38 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17792_msk_1.map
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Additional map: #1

Fileemd_17792_additional_1.map
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Half map: #2

Fileemd_17792_half_map_1.map
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Half map: #1

Fileemd_17792_half_map_2.map
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Sample components

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Entire : Influenza A/H7N9 polymerase in replicase-like conformation in pre...

EntireName: Influenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
Components
  • Complex: Influenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
    • Complex: Polymerase acidic protein, RNA-directed RNA polymerase catalytic subunit and Polymerase basic protein 2
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: 51-mer vRNA loop (v51_mut_S)
      • RNA: 51-mer vRNA loop (v51_mut_S)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Influenza A/H7N9 polymerase in replicase-like conformation in pre...

SupramoleculeName: Influenza A/H7N9 polymerase in replicase-like conformation in pre-initiation state with Pol II pS5 CTD peptide mimic bound in site 1A/2A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4

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Supramolecule #2: Polymerase acidic protein, RNA-directed RNA polymerase catalytic ...

SupramoleculeName: Polymerase acidic protein, RNA-directed RNA polymerase catalytic subunit and Polymerase basic protein 2
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))

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Supramolecule #3: 51-mer vRNA loop (v51_mut_S)

SupramoleculeName: 51-mer vRNA loop (v51_mut_S) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))

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Macromolecule #1: Polymerase acidic protein

MacromoleculeName: Polymerase acidic protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 84.394117 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MHHHHHHHHG SGSMEDFVRQ CFNPMIVELA EKAMKEYGED PKIETNKFAS ICTHLEVCFM YSDFHFIDER GESTIIESGD PNVLLKHRF EIIEGRDRTM AWTVVNSICN TTGVEKPKFL PDLYDYKENR FIEIGVTRRE VHIYYLEKAN KIKSEKTHIH I FSFTGEEM ...String:
MHHHHHHHHG SGSMEDFVRQ CFNPMIVELA EKAMKEYGED PKIETNKFAS ICTHLEVCFM YSDFHFIDER GESTIIESGD PNVLLKHRF EIIEGRDRTM AWTVVNSICN TTGVEKPKFL PDLYDYKENR FIEIGVTRRE VHIYYLEKAN KIKSEKTHIH I FSFTGEEM ATKADYTLDE ESRARIKTRL FTIRQEMASR GLWDSFRQSE RGEETIEERF EITGTMRRLA DQSLPPNFSS LE NFRAYVD GFEPNGCIEG KLSQMSKEVN ARIEPFLRTT PRPLRLPDGP PCSQRSKFLL MDALALSIED PSHEGEGIPL YDA IKCMKT FFGWKEPNII KPHEKGINPN YLLTWKQVLA ELQDIENEEK IPRTKNMKKT SQLKWALGEN MAPEKVDFED CKDV NDLKQ YDSDEPEPRS LACWIQSEFN KACELTDSSW VELDEIGEDV APIEHIASMR RNYFTAEVSH CRATEYIMKG VYINT ALLN ASCAAMDDFQ LIPMISKRRT KEGRRKTNLY GFIIKGRSHL RNDTDVVNFV SMEFSLTDPR LEPHKWEKYC VLEIGD MLL RTAVGQVSRP MFLYVRTNGT SKIKMKWGME MRRCLLQSLQ QIESMIEAES SVKEKDLTKE FFENKSETWP IGESPKG VE EGSIGKVCRT LLAKSVFNSL YASPQLEGFS AESRKLLLIV QALRDNLEPG TFDLEGLYEA IEECLINDPW VLLNASWF N SFLTHALR

UniProtKB: Polymerase acidic protein

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Macromolecule #2: RNA-directed RNA polymerase catalytic subunit

MacromoleculeName: RNA-directed RNA polymerase catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 86.496156 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDVNPTLLFL KVPVQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHKYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEIVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK ...String:
MDVNPTLLFL KVPVQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHKYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEIVQQTRV DKLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK DVMDSMDKEE MEITTHFQRK RRVRDNMTKK MVTQRTIGKK KQRLNKRSYL IRALTLNTMT KDAERGKLKR RA IATPGMQ IRGFVYFVEA LARSICEKLE QSGLPVGGNE KKAKLANVVR KMMTNSQDTE LSFTITGDNT KWNENQNPRM FLA MITYIT RNQPEWFRNV LSIAPIMFSN KMARLGKGYM FESKSMKLRT QVPAEMLANI DLKYFNKSTR EKIEKIRPLL IDGT ASLSP GMMMGMFNML STVLGVSILN LGQKKYTKTT YWWDGLQSSD DFALIVNAPN HEGIQAGVDR FYRTCKLVGI NMSKK KSYI NRTGTFEFTS FFYRYGFVAN FSMELPSFGV SGINESADMS VGVTVIKNNM INNDLGPATA QMALQLFIKD YRYTYR CHR GDTQIQTRRA FELKKLWEQT RSKAGLLVSD GGPNLYNIRN LHIPEVCLKW ELMDEDYQGR LCNPMNPFVS HKEIDSV NN AVVMPAHGPA KSMEYDAVAT THSWIPKRNR SILNTSQRGI LEDEQMYQKC CNLFEKFFPS SSYRRPVGIS SMVEAMVS R ARIDARIDFE SGRIKKEEFA EIMKICSTIE ELRRQK

UniProtKB: RNA-directed RNA polymerase catalytic subunit

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Macromolecule #3: Polymerase basic protein 2

MacromoleculeName: Polymerase basic protein 2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 88.937438 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF ...String:
MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQGQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF PNEVGARILT SESQLTITKE KKKELQDCKI APLMVAYMLE RELVRKTRFL PVAGGTSSVY IEVLHLTQGT CW EQMYTPG GEVRNDDVDQ SLIIAARNIV RRATVSADPL ASLLEMCHST QIGGVRMVDI LRQNPTEEQA VDICKAAMGL RIS SSFSFG GFTFKRTSGS SVKREEEVLT GNLQTLKIRV HEGYEEFTMV GRRATAILRK ATRRLIQLIV SGKDEQSIAE AIIV AMVFS QEDCMIKAVR GDLNFVNRAN QRLNPMHQLL RHFQKDAKVL FQNWGIEPID NVMGMIGILP DMTPSTEMSL RGVRV SKMG VDEYSSTERV VVSIDRFLRV RDQRGNVLLS PEEVSETQGT EKLTITYSSS MMWEINGPES VLVNTYQWII RNWENV KIQ WSQDPTMLYN KMEFEPFQSL VPKAARGQYS GFVRVLFQQM RDVLGTFDTV QIIKLLPFAA APPEQSRMQF SSLTVNV RG SGMRIVVRGN SPVFNYNKAT KRLTVLGKDA GALMEDPDEG TAGVESAVLR GFLILGKENK RYGPALSINE LSNLAKGE K ANVLIGQGDV VLVMKRKRDS SILTDSQTAT KRIRMAINGW SHPQFEKGGG SGGGSGGSAW SHPQFEK

UniProtKB: Polymerase basic protein 2

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Macromolecule #4: 51-mer vRNA loop (v51_mut_S)

MacromoleculeName: 51-mer vRNA loop (v51_mut_S) / type: rna / ID: 4 / Number of copies: 4
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 16.093369 KDa
SequenceString:
AGUAGAAACA AGGGUGUAUU UUCCCCUCUU UUUGUUUCCC CUGCUUUUGC U

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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

Concentration0.2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (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 / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 278761
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3) / Software - details: Ab-initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7z4o


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-8poh:
Influenza A/H7N9 polymerase symmetric dimer bound to the promoter (PA K289A/C489R)

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