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Yorodumi- PDB-6qwl: Influenza B virus (B/Panama/45) polymerase Hetermotrimer in compl... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6qwl | ||||||||||||
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Title | Influenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter | ||||||||||||
Components |
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Keywords | RNA BINDING PROTEIN / Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP | ||||||||||||
Function / homology | Function and homology information virion component => GO:0044423 / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / cap snatching / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication ...virion component => GO:0044423 / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / cap snatching / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription / nucleotide binding / host cell nucleus / RNA binding / metal ion binding Similarity search - Function | ||||||||||||
Biological species | Influenza B virus Influenza A virus | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å | ||||||||||||
Authors | Keown, J.R. / Carrique, L. / 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 | 6qwl.cif.gz | 270.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6qwl.ent.gz | 196.1 KB | Display | PDB format |
PDBx/mmJSON format | 6qwl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6qwl_validation.pdf.gz | 878.8 KB | Display | wwPDB validaton report |
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Full document | 6qwl_full_validation.pdf.gz | 896.7 KB | Display | |
Data in XML | 6qwl_validation.xml.gz | 44.5 KB | Display | |
Data in CIF | 6qwl_validation.cif.gz | 68.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/qw/6qwl ftp://data.pdbj.org/pub/pdb/validation_reports/qw/6qwl | HTTPS FTP |
-Related structure data
Related structure data | 4660MC 4661C 4663C 4664C 4666C 4986C 6qnwC 6qpfC 6qpgC 6qx3C 6qx8C 6qxeC 6rr7C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 83161.055 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990) Gene: PA / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: O36432, Hydrolases; Acting on ester bonds |
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#2: Protein | Mass: 84378.180 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990) Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O36430, RNA-directed RNA polymerase |
#3: Protein | Mass: 89097.453 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990) Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O36431 |
#4: RNA chain | Mass: 4683.753 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus |
#5: RNA chain | Mass: 4531.827 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus |
-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 | ||||||||||||||||||||||||||||
Source (natural) |
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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 | ||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1 | ||||||||||||||||||||||||||||
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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 4.38 sec. / Electron dose: 1.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4711 |
Image scans | Movie frames/image: 28 / Used frames/image: 1-28 |
-Processing
Software |
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1012085 / Details: template picking in cryosparc v2.5 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1012085 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 57 / Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5EPI Accession code: 5EPI / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||
Refinement | Stereochemistry target values: CDL v1.2 | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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