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Yorodumi- PDB-6qxe: Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrime... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6qxe | ||||||||||||
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Title | Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205 | ||||||||||||
Components |
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Keywords | RNA BINDING PROTEIN / Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP | ||||||||||||
Function / homology | Function and homology information 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds ...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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / 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 | Influenza A virus | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.15 Å | ||||||||||||
Authors | Carrique, L. / Keown, J.R. / Fan, H. / Fodor, E. / Grimes, J.M. | ||||||||||||
Funding support | United Kingdom, 3items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6qxe.cif.gz | 871.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6qxe.ent.gz | 726.2 KB | Display | PDB format |
PDBx/mmJSON format | 6qxe.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qx/6qxe ftp://data.pdbj.org/pub/pdb/validation_reports/qx/6qxe | HTTPS FTP |
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-Related structure data
Related structure data | 4666MC 4660C 4661C 4663C 4664C 4986C 6qnwC 6qpfC 6qpgC 6qwlC 6qx3C 6qx8C 6rr7C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 14835.375 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) Production host: Spodoptera frugiperda (fall armyworm) #2: Protein | Mass: 83100.797 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) Gene: PA / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: P03434, Hydrolases; Acting on ester bonds #3: Protein | Mass: 86524.086 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03432, RNA-directed RNA polymerase #4: Protein | Mass: 86163.391 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza A virus (A/nt/60/1968(H3N2)) Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03429 #5: RNA chain | Mass: 4901.097 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Influenza A virus (A/nt/60/1968(H3N2)) |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight | Value: 0.25 MDa / Experimental value: NO | ||||||||||||||||||||||||||||
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Source (recombinant) |
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Buffer solution | pH: 7.5 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. | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: blot for 3.5 sec before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 6 sec. / Electron dose: 1.25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2456 |
EM imaging optics | Phase plate: VOLTA PHASE PLATE |
Image scans | Movie frames/image: 24 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 406945 / Details: template picking in cryosparc v2.5 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27861 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 95 / Protocol: RIGID BODY FIT / Space: REAL / Details: Rigid body fit only | ||||||||||||||||||||||||||||||||||||
Atomic model building |
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