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- EMDB-4660: Influenza B virus (B/Panama/45) polymerase Hetermotrimer in compl... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4660 | ||||||||||||
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Title | Influenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter | ||||||||||||
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![]() | Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP / RNA BINDING PROTEIN | ||||||||||||
Function / homology | ![]() virion component => GO:0044423 / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication ...virion component => GO:0044423 / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / 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 | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.1 Å | ||||||||||||
![]() | Keown JR / Carrique L | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 60 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.2 KB 22.2 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.2 KB | Display | ![]() |
Images | ![]() | 33 KB | ||
Filedesc metadata | ![]() | 8.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6qwlMC ![]() 4661C ![]() 4663C ![]() 4664C ![]() 4666C ![]() 4986C ![]() 6qnwC ![]() 6qpfC ![]() 6qpgC ![]() 6qx3C ![]() 6qx8C ![]() 6qxeC ![]() 6rr7C M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...
Entire | Name: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter |
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Components |
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-Supramolecule #1: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...
Supramolecule | Name: 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. |
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Molecular weight | Theoretical: 250 KDa |
-Supramolecule #2: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...
Supramolecule | Name: 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. |
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Source (natural) | Organism: ![]() |
-Supramolecule #3: Influenza B (Panama/45) polymerase Hetermotrimer in complex with ...
Supramolecule | Name: 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. |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Polymerase acidic protein
Macromolecule | Name: Polymerase acidic protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 83.161055 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: Polymerase acidic protein |
-Macromolecule #2: RNA-directed RNA polymerase catalytic subunit
Macromolecule | Name: RNA-directed RNA polymerase catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 84.37818 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: RNA-directed RNA polymerase catalytic subunit |
-Macromolecule #3: Polymerase basic protein 2
Macromolecule | Name: Polymerase basic protein 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 89.097453 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: Polymerase basic protein 2 |
-Macromolecule #4: 3' cRNA
Macromolecule | Name: 3' cRNA / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 4.683753 KDa |
Sequence | String: GGCCUUGUUU CUACU |
-Macromolecule #5: 5' cRNA
Macromolecule | Name: 5' cRNA / type: rna / ID: 5 / Number of copies: 1 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 4.531827 KDa |
Sequence | String: AGCAAAAGCA GGCC |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.35 mg/mL | |||||||||
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Buffer | pH: 7.5 / Component:
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. | |||||||||
Grid | Model: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa | |||||||||
Vitrification | Cryogen 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
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |