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- EMDB-19368: Influenza polymerase A/H7N9-4M replication complex, an asymmetric... -

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Basic information

Entry
Database: EMDB / ID: EMD-19368
TitleInfluenza polymerase A/H7N9-4M replication complex, an asymmetric polymerase dimer bound to human ANP32A
Map data
Sample
  • Complex: Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and one FluPolA/H7N9-4M encapsidase, bridged by human ANP32A
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • Protein or peptide: Acidic leucine-rich nuclear phosphoprotein 32 family member A
  • Ligand: MAGNESIUM ION
KeywordsViral polymerase / VIRAL PROTEIN
Function / homology
Function and homology information


HuR (ELAVL1) binds and stabilizes mRNA / nucleocytoplasmic transport / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / virion component / histone binding / regulation of apoptotic process ...HuR (ELAVL1) binds and stabilizes mRNA / nucleocytoplasmic transport / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / virion component / histone binding / regulation of apoptotic process / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / intracellular signal transduction / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / perinuclear region of cytoplasm / endoplasmic reticulum / RNA binding / nucleoplasm / nucleus / metal ion binding / cytoplasm
Similarity search - Function
Acidic leucine-rich nuclear phosphoprotein 32 / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : ...Acidic leucine-rich nuclear phosphoprotein 32 / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / 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. / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
Polymerase acidic protein / Acidic leucine-rich nuclear phosphoprotein 32 family member A / 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: 3.25 Å
AuthorsArragain B / Cusack S
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-18-CE18-0028 France
CitationJournal: Nat Commun / Year: 2024
Title: Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.
Authors: Benoît Arragain / Tim Krischuns / Martin Pelosse / Petra Drncova / Martin Blackledge / Nadia Naffakh / Stephen Cusack /
Abstract: Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal ...Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.
History
DepositionJan 8, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19368.png

Downloads & links

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Map

FileDownload / File: emd_19368.map.gz / Format: CCP4 / Size: 282.6 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 420 pix.
= 352.8 Å		0.84 Å/pix.
x 420 pix.
= 352.8 Å		0.84 Å/pix.
x 420 pix.
= 352.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.9025009 - 1.6426784
Average (Standard dev.)0.0026661796 (±0.039797135)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 352.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19368_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and on...

EntireName: Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and one FluPolA/H7N9-4M encapsidase, bridged by human ANP32A
Components
  • Complex: Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and one FluPolA/H7N9-4M encapsidase, bridged by human ANP32A
    • Protein or peptide: Polymerase acidic protein
    • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
    • Protein or peptide: Polymerase basic protein 2
    • Protein or peptide: Acidic leucine-rich nuclear phosphoprotein 32 family member A
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and on...

SupramoleculeName: Asymmetric dimer composed of one FluPolA/H7N9-4M replicase and one FluPolA/H7N9-4M encapsidase, bridged by human ANP32A
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#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: 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: 2 / 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: 2 / 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 #4: Acidic leucine-rich nuclear phosphoprotein 32 family member A

MacromoleculeName: Acidic leucine-rich nuclear phosphoprotein 32 family member A
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.747785 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKHHHHHHPM SDYDIPTTEN LYFQGAMEMG RRIHLELRNR TPSDVKELVL DNSRSNEGKL EGLTDEFEEL EFLSTINVGL TSIANLPKL NKLKKLELSD NRVSGGLEVL AEKCPNLTHL NLSGNKIKDL STIEPLKKLE NLKSLDLFNC EVTNLNDYRE N VFKLLPQL ...String:
MKHHHHHHPM SDYDIPTTEN LYFQGAMEMG RRIHLELRNR TPSDVKELVL DNSRSNEGKL EGLTDEFEEL EFLSTINVGL TSIANLPKL NKLKKLELSD NRVSGGLEVL AEKCPNLTHL NLSGNKIKDL STIEPLKKLE NLKSLDLFNC EVTNLNDYRE N VFKLLPQL TYLDGYDRDD KEAPDSDAEG YVEGLDDEEE DEDEEEYDED AQVVEDEEDE DEEEEGEEED VSGEEEEDEE GY NDGEVDD EEDEEELGEE ERGQKRKREP EDEGEDDD

UniProtKB: Acidic leucine-rich nuclear phosphoprotein 32 family member A

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS 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: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 18596
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-8rmr:
Influenza polymerase A/H7N9-4M replication complex, an asymmetric polymerase dimer bound to human ANP32A

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