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Yorodumi- PDB-8aox: CryoEM structure of the Chikungunya virus nsP1 capping pores in c... -
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-Basic information
Entry | Database: PDB / ID: 8aox | ||||||
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Title | CryoEM structure of the Chikungunya virus nsP1 capping pores in complex with SAM | ||||||
Components | mRNA-capping enzyme nsP1 | ||||||
Keywords | VIRAL PROTEIN / Alphavirus Replication complex Capping pores Membrane pore Methyltransferase gunayltransferase VIRAL PROTEIN | ||||||
Function / homology | Function and homology information host cell filopodium / ADP-ribose 1''-phosphate phosphatase / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide 5'-phosphatase activity / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity / regulation of cytoskeleton organization / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity ...host cell filopodium / ADP-ribose 1''-phosphate phosphatase / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide 5'-phosphatase activity / polynucleotide adenylyltransferase / poly(A) RNA polymerase activity / regulation of cytoskeleton organization / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / cysteine-type peptidase activity / Transferases; Transferring one-carbon groups; Methyltransferases / host cell cytoplasmic vesicle membrane / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nucleoside-triphosphate phosphatase / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / RNA helicase / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell nucleus / GTP binding / host cell plasma membrane / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / membrane / metal ion binding Similarity search - Function | ||||||
Biological species | Chikungunya virus strain S27-African prototype | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||
Authors | Jones, R. / Hons, M. / Reguera, J. | ||||||
Funding support | France, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores. Authors: Rhian Jones / Michael Hons / Nadia Rabah / Noelia Zamarreño / Rocío Arranz / Juan Reguera / Abstract: Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is ...Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an mGMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and mGTP in the active site, and the subsequent covalent transfer of mGMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8aox.cif.gz | 2.6 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8aox.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 8aox.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ao/8aox ftp://data.pdbj.org/pub/pdb/validation_reports/ao/8aox | HTTPS FTP |
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-Related structure data
Related structure data | 15555MC 8aovC 8aowC 8apxC 8axvC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 59975.422 Da / Num. of mol.: 24 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Chikungunya virus strain S27-African prototype Strain: S27-African prototype / Production host: Baculovirus expression vector pFastBac1-HM References: UniProt: Q8JUX6, Transferases; Transferring one-carbon groups; Methyltransferases, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases #2: Chemical | ChemComp-ZN / #3: Chemical | ChemComp-SAM / #4: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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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
Component | Name: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (SAM) ligand. Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Value: 1.2 MDa / Experimental value: YES | |||||||||||||||||||||||||
Source (natural) | Organism: Chikungunya virus strain S27-African prototype | |||||||||||||||||||||||||
Source (recombinant) | Organism: Baculovirus expression vector pFastBac1-HM | |||||||||||||||||||||||||
Buffer solution | pH: 7.6 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Specimen was incubated with 0.5mM SAM | |||||||||||||||||||||||||
Specimen support | Grid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K |
-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 defocus max: 2500 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 70 µm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 4 sec. / Electron dose: 42.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4500 |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 366156 | ||||||||||||||||||||||||
Symmetry | Point symmetry: D12 (2x12 fold dihedral) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69057 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 6Z0V | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 37.4 Å2 | ||||||||||||||||||||||||
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