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- EMDB-4986: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to... -

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Basic information

Entry
Database: EMDB / ID: EMD-4986
TitleInfluenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and capped RNA primer
Map dataNone
Sample
  • Complex: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and Capped RNA
    • Complex: polymerase Heterotrimer
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Capped messenger RNA
      • RNA: RNA (5'-D(*(M7G))-R(P*AP*AP*UP*CP*U)-3')
    • Complex: Influenza A 5' and 3' viral RNA promoter
      • RNA: RNA (5'-R(P*GP*CP*CP*UP*GP*CP*UP*UP*UP*UP*G)-3')
      • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*C)-3')
KeywordsInfluenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP / RNA BINDING 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm ...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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / 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 basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus (A/nt/60/1968(H3N2)) / Spodoptera frugiperda (fall armyworm) / Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsCarrique L / Keown JR
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
Authors: Fan H / Walker AP / Carrique L / Keown JR / Serna Martin I / Karia D / Sharps J / Hengrung N / Pardon E / Steyaert J / Grimes JM / Fodor E
History
DepositionMay 17, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseSep 4, 2019-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rr7
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_4986.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.085 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.25079343 - 0.46317026
Average (Standard dev.)0.00009071167 (±0.009107879)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 271.25 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0851.0851.085
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z271.250271.250271.250
α/β/γ90.00090.00090.000
start NX/NY/NZ-39-149-104
NX/NY/NZ201201211
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.2510.4630.000

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Supplemental data

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Sample components

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Entire : Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to...

EntireName: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and Capped RNA
Components
  • Complex: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and Capped RNA
    • Complex: polymerase Heterotrimer
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Capped messenger RNA
      • RNA: RNA (5'-D(*(M7G))-R(P*AP*AP*UP*CP*U)-3')
    • Complex: Influenza A 5' and 3' viral RNA promoter
      • RNA: RNA (5'-R(P*GP*CP*CP*UP*GP*CP*UP*UP*UP*UP*G)-3')
      • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*C)-3')

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Supramolecule #1: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to...

SupramoleculeName: Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and Capped RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.
Molecular weightTheoretical: 250 KDa

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Supramolecule #2: polymerase Heterotrimer

SupramoleculeName: polymerase Heterotrimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))

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Supramolecule #3: Capped messenger RNA

SupramoleculeName: Capped messenger RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #4: Influenza A 5' and 3' viral RNA promoter

SupramoleculeName: Influenza A 5' and 3' viral RNA promoter / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))

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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 / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2
Molecular weightTheoretical: 83.100797 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEDFVRQCFN PMIVELAEKA MKEYGEDLKI ETNKFAAICT HLEVCFMYSD FHFINEQGES IVVELDDPNA LLKHRFEIIE GRDRTMAWT VVNSICNTTG AEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SENTHIHIFS FTGEEMATKA D YTLDEESR ...String:
MEDFVRQCFN PMIVELAEKA MKEYGEDLKI ETNKFAAICT HLEVCFMYSD FHFINEQGES IVVELDDPNA LLKHRFEIIE GRDRTMAWT VVNSICNTTG AEKPKFLPDL YDYKENRFIE IGVTRREVHI YYLEKANKIK SENTHIHIFS FTGEEMATKA D YTLDEESR ARIKTRLFTI RQEMANRGLW DSFRQSERGE ETIEERFEIT GTMRRLADQS LPPNFSCLEN FRAYVDGFEP NG YIEGKLS QMSKEVNAKI EPFLKTTPRP IRLPDGPPCF QRSKFLLMDA LKLSIEDPSH EGEGIPLYDA IKCMRTFFGW KEP YIVKPH EKGINPNYLL SWKQVLAELQ DIENEEKIPR TKNMKKTSQL KWALGENMAP EKVDFDNCRD VSDLKQYDSD EPEL RSLSS WIQNEFNKAC ELTDSTWIEL DEIGEDVAPI EYIASMRRNY FTAEVSHCRA TEYIMKGVYI NTALLNASCA AMDDF QLIP MISKCRTKEG RRKTNLYGFI IKGRSHLRND TDVVNFVSME FSLTDPRLEP HKWEKYCVLE IGDMLLRSAI GQMSRP MFL YVRTNGTSKI KMKWGMEMRR CLLQSLQQIE SMIEAESSVK EKDMTKEFFE NKSETWPIGE SPKGVEDGSI GKVCRTL LA KSVFNSLYAS PQLEGFSAES RKLLLVVQAL 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 / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2
Molecular weightTheoretical: 86.524086 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDVNPTLLFL KVPAQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHQYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEVVQQTRV DRLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK ...String:
MDVNPTLLFL KVPAQNAIST TFPYTGDPPY SHGTGTGYTM DTVNRTHQYS EKGKWTTNTE TGAPQLNPID GPLPEDNEPS GYAQTDCVL EAMAFLEESH PGIFENSCLE TMEVVQQTRV DRLTQGRQTY DWTLNRNQPA ATALANTIEV FRSNGLTANE S GRLIDFLK DVMESMDKEE MEITTHFQRK RRVRDNMTKK MVTQRTIGKK KQRVNKRSYL IRALTLNTMT KDAERGKLKR RA IATPGMQ IRGFVYFVET LARSICEKLE QSGLPVGGNE KKAKLANVVR KMMTNSQDTE LSFTITGDNT KWNENQNPRM FLA MITYIT KNQPEWFRNV LSIAPIMFSN KMARLGKGYM FESKSMKLRT QIPAEMLASI DLKYFNESTR KKIEKIRPLL IDGT ASLSP GMMMGMFNML STVLGVSILN LGQKRYTKTT YWWDGLQSSD DFALIVNAPN HEGIQAGVDR FYRTCKLVGI NMSKK KSYI NRTGTFEFTS FFYRYGFVAN FSMELPSFGV SGINESADMS IGVTVIKNNM INNDLGPATA QMALQLFIKD YRYTYR CHR GDTQIQTRRS FELKKLWEQT RSKAGLLVSD GGPNLYNIRN LHIPEVCLKW ELMDEDYQGR LCNPLNPFVS HKEIESV 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 (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2
Molecular weightTheoretical: 86.958242 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MERIKELRNL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPSLRMKWM MAMKYPITAD KRITEMVPER NEQGQTLWSK MSDAGSDRV MVSPLAVTWW NRNGPMTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF ...String:
MERIKELRNL MSQSRTREIL TKTTVDHMAI IKKYTSGRQE KNPSLRMKWM MAMKYPITAD KRITEMVPER NEQGQTLWSK MSDAGSDRV MVSPLAVTWW NRNGPMTSTV HYPKVYKTYF EKVERLKHGT FGPVHFRNQV KIRRRVDINP GHADLSAKEA Q DVIMEVVF PNEVGARILT SESQLTITKE KKEELQDCKI SPLMVAYMLE RELVRKTRFL PVAGGTSSVY IEVLHLTQGT CW EQMYTPG GEVRNDDVDQ SLIIAARNIV RRAAVSADPL ASLLEMCHST QIGGTRMVDI LRQNPTEEQA VDICKAAMGL RIS SSFSFG GFTFKRTSGS SIKREEELLT GNLQTLKIRV HDGYEEFTMV GKRATAILRK ATRRLVQLIV SGRDEQSVAE AIIV AMVFS QEDCMIKAVR GDLNFVNRAN QRLNPMHQLL RHFQKDAKVL FQNWGIEHID NVMGMIGVLP DMTPSTEMSM RGIRV SKMG VDEYSSTERV VVSIDRFLRV RDQRGNVLLS PEEVSETQGT EKLTITYSSS MMWEINGPES VLVNTYQWII RNWETV KIQ WSQNPTMLYN KMEFEPFQSL VPKAIRGQYS GFVRTLFQQM RDVLGTFDTT QIIKLLPFAA APPKQSRMQF SSLTVNV RG SGMRILVRGN SPAFNYNKTT KRLTILGKDA GTLIEDPDEG TSGVESAVLR GFLILGKEDR RYGPALSINE LSNLAKGE K ANVLIGQGDV VLVMKRKRDS SILTDSQTAT KRIRMAINEN LYFQ

UniProtKB: Polymerase basic protein 2

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Macromolecule #4: RNA (5'-D(*(M7G))-R(P*AP*AP*UP*CP*U)-3')

MacromoleculeName: RNA (5'-D(*(M7G))-R(P*AP*AP*UP*CP*U)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 1.971188 KDa
SequenceString:
(M7G)AAUCU

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Macromolecule #5: RNA (5'-R(P*GP*CP*CP*UP*GP*CP*UP*UP*UP*UP*G)-3')

MacromoleculeName: RNA (5'-R(P*GP*CP*CP*UP*GP*CP*UP*UP*UP*UP*G)-3') / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))
Molecular weightTheoretical: 4.393587 KDa
SequenceString:
GGCCUGCUUU UGCU

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Macromolecule #6: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*C)-3')

MacromoleculeName: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*C)-3')
type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))
Molecular weightTheoretical: 4.862017 KDa
SequenceString:
AGUAGAAACA AGGCC

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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

Concentration0.35 mg/mL
BufferpH: 7.5 / Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMNaCl

Details: Sample was purified in 20 mM HEPES, pH 7.5, 150 mM NaCl with Tween 20 added to a final concentration 0f 0.005% prior to plunging grids.
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 3.5 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 6203 / Average exposure time: 2.0 sec. / Average electron dose: 36.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 0.7000000000000001 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2210168 / Details: template picking in cryosparc v2.5
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model was generated by cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 170144
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 10 / Avg.num./class: 45000 / Software - Name: RELION (ver. 3)

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Atomic model buiding 1

Initial modelPDB ID:

6qnw


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 65
Output model

PDB-6rr7:
Influenza A virus (A/NT/60/1968) polymerase Heterotrimer bound to 3'5' vRNA promoter and capped RNA primer

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