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- EMDB-18871: Influenza A/H7N9 polymerase in self-stalled pre-termination state... -

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Basic information

Entry
Database: EMDB / ID: EMD-18871
TitleInfluenza A/H7N9 polymerase in self-stalled pre-termination state, with Pol II pS5 CTD peptide mimic bound in site 1A/2A.
Map data
Sample
  • Complex: Heterotrimeric influenza polymerase
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 51-mer vRNA loop (v51_mut_S)
    • RNA: RNA product
    • Protein or peptide: RNA Pol II CTD 6 repeats (site 1A/2A)
  • Ligand: PYROPHOSPHATE 2-
  • 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 / symbiont-mediated suppression of host gene expression ...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 / 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)) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.43 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
DepositionNov 9, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18871.png

Downloads & links

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Map

FileDownload / File: emd_18871.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 380 pix.
= 333.64 Å		0.88 Å/pix.
x 380 pix.
= 333.64 Å		0.88 Å/pix.
x 380 pix.
= 333.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.878 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.5817887 - 1.0567762
Average (Standard dev.)0.0005415717 (±0.018472519)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 333.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18871_msk_1.map
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Additional map: #1

Fileemd_18871_additional_1.map
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Half map: #1

Fileemd_18871_half_map_1.map
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Half map: #2

Fileemd_18871_half_map_2.map
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Sample components

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Entire : Heterotrimeric influenza polymerase

EntireName: Heterotrimeric influenza polymerase
Components
  • Complex: Heterotrimeric influenza polymerase
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • RNA: 51-mer vRNA loop (v51_mut_S)
    • RNA: RNA product
    • Protein or peptide: RNA Pol II CTD 6 repeats (site 1A/2A)
  • Ligand: PYROPHOSPHATE 2-
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Heterotrimeric influenza polymerase

SupramoleculeName: Heterotrimeric influenza polymerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
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: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 82.829578 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEDFVRQCFN PMIVELAEKA MKEYGEDPKI ETNKFASICT HLEVCFMYSD FHFIDERGES TIIESGDPNV LLKHRFEIIE GRDRTMAWT VVNSICNTTG VEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SEKTHIHIFS FTGEEMATKA D YTLDEESR ...String:
MEDFVRQCFN PMIVELAEKA MKEYGEDPKI ETNKFASICT HLEVCFMYSD FHFIDERGES TIIESGDPNV LLKHRFEIIE GRDRTMAWT VVNSICNTTG VEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SEKTHIHIFS FTGEEMATKA D YTLDEESR ARIKTRLFTI RQEMASRGLW DSFRQSERGE ETIEERFEIT GTMRRLADQS LPPNFSSLEN FRAYVDGFEP NG CIEGKLS QMSKEVNARI EPFLRTTPRP LRLPDGPPCS QRSKFLLMDA LKLSIEDPSH EGEGIPLYDA IKCMKTFFGW KEP NIIKPH EKGINPNYLL TWKQVLAELQ DIKNEEKIPR TKNMKKTSQL KWALGENMAP EKVDFEDCKD VNDLKQYDSD EPEP RSLAC WIQSEFNKAC ELTDSSWVEL DEIGEDVAPI EHIASMRRNY FTAEVSHCRA TEYIMKGVYI NTALLNASCA AMDDF QLIP MISKCITKEG RRKTNLYGFI IKGRSHLRND TDVVNFVSME FSLTDPRLEP HKWEKYCVLE IGDMLLRTAV GQVSRP MFL YVRTNGTSKI KMKWGMEMRR CLLQSLQQIE SMIEAESSVK EKDLTKEFFE NKSETWPIGE SPKGVEEGSI GKVCRTL LA KSVFNSLYAS PQLEGFSAES RKLLLIVQAL RDNLEPGTFD LEGLYEAIEE CLINDPWVLL NASWFNSFLT HALR

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: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 86.424031 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 FELGKLWEQT 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: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 89.037578 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQRQTLWSK TNDAGSDRV MVSPLAVTWW NRNGPTTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF ...String:
MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPALRMKWM MAMKYPITAD KRIMEMIPER NEQRQTLWSK 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 #6: RNA Pol II CTD 6 repeats (site 1A/2A)

MacromoleculeName: RNA Pol II CTD 6 repeats (site 1A/2A) / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.816331 KDa
SequenceString:
YSPT(SEP)PSYSP T(SEP)PSYSPT(SEP)P SYSPT(SEP)PSYS PT(SEP)PSYSPT(SEP) PS

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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: 1
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: RNA product

MacromoleculeName: RNA product / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Molecular weightTheoretical: 11.228926 KDa
SequenceString:
GCAAAAGCAG GGGAAACAAA AAGAGGGGAA AAUA

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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-

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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

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 reconstructionResolution.type: BY AUTHOR / Resolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 29072
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8r3k:
Influenza A/H7N9 polymerase in self-stalled pre-termination state, with Pol II pS5 CTD peptide mimic bound in site 1A/2A.

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