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- EMDB-4660: Influenza B virus (B/Panama/45) polymerase Hetermotrimer in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-4660
TitleInfluenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
Map dataNone
Sample
  • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
    • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
      • RNA: 3' cRNA
      • RNA: 5' cRNA
Function / homology
Function and homology information


virion component => GO:0044423 / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication ...virion component => GO:0044423 / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA polymerase PB2 CAP binding domain / Polymerase acidic protein / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA
Similarity search - Domain/homology
RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2 / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza B virus (B/Panama/45/1990) / Influenza A virus / Influenza B virus (strain B/Panama/45/1990)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKeown JR / Carrique L / Fan H / Fodor E / Grimes JM
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
Authors: Haitian Fan / Alexander P Walker / Loïc Carrique / Jeremy R Keown / Itziar Serna Martin / Dimple Karia / Jane Sharps / Narin Hengrung / Els Pardon / Jan Steyaert / Jonathan M Grimes / Ervin Fodor /
Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented ...Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol) composed of PB1, PB2 and PA subunits. Although the high-resolution crystal structure of FluPol of bat influenza A virus has previously been reported, there are no complete structures available for human and avian FluPol. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol that could be targeted in the development of antiviral drugs.
History
DepositionMar 5, 2019-
Header (metadata) releaseMar 13, 2019-
Map releaseSep 4, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qwl
  • Surface level: 5.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4660.png

Downloads & links

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Map

FileDownload / File: emd_4660.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 5.6 / Movie #1: 5.6
Minimum - Maximum-25.019184 - 38.01063
Average (Standard dev.)-0.00000000001 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
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/NS256256256
D min/max/mean-25.01938.011-0.000

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Supplemental data

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Sample components

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Entire : Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...

EntireName: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
Components
  • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
    • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
      • Protein or peptide: Polymerase acidic protein
      • Protein or peptide: RNA-directed RNA polymerase catalytic subunit
      • Protein or peptide: Polymerase basic protein 2
    • Complex: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
      • RNA: 3' cRNA
      • RNA: 5' cRNA

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Supramolecule #1: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...

SupramoleculeName: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
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: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...

SupramoleculeName: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
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.
Source (natural)Organism: Influenza B virus (B/Panama/45/1990)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...

SupramoleculeName: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
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.
Source (natural)Organism: Influenza A virus
Recombinant expressionOrganism: synthetic construct (others)

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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 B virus (strain B/Panama/45/1990)
Molecular weightTheoretical: 83.161055 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDTFITRNFQ TTIIQKAKNT MAEFSEDPEL QPAMLFNICV HLEVCYVISD MNFLDEEGKS YTALEGQGKE QNLRPQYEVI EGMPRTIAW MVQRSLAQEH GIETPKYLAD LFDYKTKRFI EVGITKGLAD DYFWKKKEKL GNSMELMIFS YNQDYSLSNE S SLDEEGKG ...String:
MDTFITRNFQ TTIIQKAKNT MAEFSEDPEL QPAMLFNICV HLEVCYVISD MNFLDEEGKS YTALEGQGKE QNLRPQYEVI EGMPRTIAW MVQRSLAQEH GIETPKYLAD LFDYKTKRFI EVGITKGLAD DYFWKKKEKL GNSMELMIFS YNQDYSLSNE S SLDEEGKG RVLSRLTELQ AELSLKNLWQ VLIGEEDVEK GIDFKLGQTI SRLRDISVPA GFSNFEGMRS YIDNIDPKGA IE RNLARMS PLVSATPKKL KWEDLRPIGP HIYNHELPEV PYNAFLLMSD ELGLANMTEG KSKKPKTLAK ECLEKYSTLR DQT DPILIM KSEKANENFL WKLWRDCVNT ISNEEMSNEL QKTNYAKWAT GDGLTYQKIM KEVAIDDETM CQEEPKIPNK CRVA AWVQT EMNLLSTLTS KRALDLPEIG PDVAPVEHVG SERRKYFVNE INYCKASTVM MKYVLFHTSL LNESNASMGK YKVIP ITNR VVNEKGESFD MLYGLAVKGQ SHLRGDTDVV TVVTFEFSST DPRVDSGKWP KYTVFRIGSL FVSGREKSVY LYCRVN GTN KIQMKWGMEA RRCLLQSMQQ MEAIVEQESS IQGYDMTKAC FKGDRVNSPK TFSIGTQEGK LVKGSFGKAL RVIFTKC LM HYVFGNAQLE GFSAESRRLL LLIQALKDRK GPWVFDLEGM YSGIEECISN NPWVIQSAYW FNEWLGFEKE GSKVLESV D EIMDE

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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 B virus (strain B/Panama/45/1990)
Molecular weightTheoretical: 84.37818 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNINPYFLFI DVPIQAAIST TFPYTGVPPY SHGTGTGHTI DTVIRTHEYS NKGKQYVSDV TGCTMVDPTN GPLPEDNEPS AYAQLDCVL EALDRMDEEH PGLFQAASQN AMEALMVTTV DKLTQGRQTF DWTVCRNQPA ATALNTTITS FRLNDLNGAD K GGLVPFCQ ...String:
MNINPYFLFI DVPIQAAIST TFPYTGVPPY SHGTGTGHTI DTVIRTHEYS NKGKQYVSDV TGCTMVDPTN GPLPEDNEPS AYAQLDCVL EALDRMDEEH PGLFQAASQN AMEALMVTTV DKLTQGRQTF DWTVCRNQPA ATALNTTITS FRLNDLNGAD K GGLVPFCQ DIIDSLDKPE MTFFSVKNIK KKLPAKNRKG FLIKRIPMKV KDRITRVEYI KRALSLNTMT KDAERGKLKR RA IATAGIQ IRGFVLVVEN LAKNICENLE QSGLPVGGNE KKAKLSNAVA KMLSNCPPGG ISMTVTGDNT KWNECLNPRI FLA MTERIT RDSPIWFRDF CSIAPVLFSN KIARLGKGFM ITSKTKRLKA QIPCPDLFSI PLERYNEETR AKLKKLKPFF NEEG TASLS PGMMMGMFNM LSTVLGVAAL GIKNIGNKEY LWDGLQSSDD FALFVNAKDE ETCMEGINDF YRTCKLLGIN MSKKK SYCN ETGMFEFTSM FYRDGFVSNF AMEIPSFGVA GVNESADMAI GMTIIKNNMI NNGMGPATAQ TAIQLFIADY RYTYKC HRG DSKVEGKRMK IIKELWENTK GRDGLLVADG GPNIYNLRNL HIPEIVLKYN LMDPEYKGRL LHPQNPFVGH LSIEGIK EA DITPAHGPVK KMDYDAVSGT HSWRTKRNRS ILNTDQRNMI LEEQCYAKCC NLFEACFNSA SYRKPVGQHS MLEAMAHR L RMDARLDYES GRMSKDDFEK AMAHLGEIGY I

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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 B virus (strain B/Panama/45/1990)
Molecular weightTheoretical: 89.097453 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTLAKIELLK QLLRDNEAKT VLKQTTVDQY NIIRKFNTSR IEKNPSLRMK WAMCSNFPLA LTKGDMANRI PLEYKGIQLK TNAEDIGTK GQMCSIAAVT WWNTYGPIGD TEGFEKVYES FFLRKMRLDN ATWGRITFGP VERVRKRVLL NPLTKEMPPD E ASNVIMEI ...String:
MTLAKIELLK QLLRDNEAKT VLKQTTVDQY NIIRKFNTSR IEKNPSLRMK WAMCSNFPLA LTKGDMANRI PLEYKGIQLK TNAEDIGTK GQMCSIAAVT WWNTYGPIGD TEGFEKVYES FFLRKMRLDN ATWGRITFGP VERVRKRVLL NPLTKEMPPD E ASNVIMEI LFPKEAGIPR ESTWIHRELI KEKREKLKGT MITPIVLAYM LERELVARRR FLPVAGATSA EFIEMLHCLQ GE NWRQIYH PGGNKLTESR SQSMIVACRK IIRRSIVASN PLELAVEIAN KTVIDTEPLK SCLTAIDGGD VACDIIRAAL GLK IRQRQR FGRLELKRIS GRGFKNDEEI LIGNGTIQKI GIWDGEEEFH VRCGECRGIL KKSKMRMEKL LINSAKKEDM KDLI ILCMV FSQDTRMFQG VRGEINFLNR AGQLLSPMYQ LQRYFLNRSN DLFDQWGYEE SPKASELHGI NELMNASDYT LKGVV VTKN VIDDFSSTET EKVSITKNLS LIKRTGEVIM GANDVSELES QAQLMITYDT PKMWEMGTTK ELVQNTYQWV LKNLVT LKA QFLLGKEDMF QWDAFEAFES IIPQKMAGQY SGFARAVLKQ MRDQEVMKTD QFIKLLPFCF SPPKLRSNGE PYQFLRL VL KGGGENFIEV RKGSPLFSYN PQTEVLTICG RMMSLKGKIE DEERNRSMGN AVLAGFLVSG KYDPDLGDFK TIEELEKL K PGEKANILLY QGKPVKVVKR KRYSALSNDI SQGIKRQRMT VESMGWALSA RENLYFQ

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Macromolecule #4: 3' cRNA

MacromoleculeName: 3' cRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 4.683753 KDa
SequenceString:
GGCCUUGUUU CUACU

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Macromolecule #5: 5' cRNA

MacromoleculeName: 5' cRNA / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 4.531827 KDa
SequenceString:
AGCAAAAGCA GGCC

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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 mMNaClSodium chloride

Details: Sample was purified in 20 mM HEPES, pH 7.5, 150 mM NaCl with Tween 20 added to a final concentration 0f 0.05% prior to plunging grids.
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-28 / Number grids imaged: 1 / Number real images: 4711 / Average exposure time: 4.38 sec. / Average electron dose: 1.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1012085 / Details: template picking in cryosparc v2.5
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model was generated by cryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.5) / Details: Cryosparc
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.5)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.5) / Number images used: 1012085
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5epi

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 57
Output model

PDB-6qwl:
Influenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter

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