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Yorodumi- PDB-8apx: CryoEM structure of the Chikungunya virus nsP1 capping pores in c... -
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-Basic information
Entry | Database: PDB / ID: 8apx | ||||||
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Title | CryoEM structure of the Chikungunya virus nsP1 capping pores in covalent complex with a 7GMP cap structure | ||||||
Components | Polyprotein P1234 | ||||||
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 / cysteine-type peptidase activity / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 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 / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / 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: 3.2 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 | 8apx.cif.gz | 1.2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8apx.ent.gz | 834.6 KB | Display | PDB format |
PDBx/mmJSON format | 8apx.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8apx_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 8apx_full_validation.pdf.gz | 2 MB | Display | |
Data in XML | 8apx_validation.xml.gz | 169.5 KB | Display | |
Data in CIF | 8apx_validation.cif.gz | 236.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ap/8apx ftp://data.pdbj.org/pub/pdb/validation_reports/ap/8apx | HTTPS FTP |
-Related structure data
Related structure data | 15578MC 8aovC 8aowC 8aoxC 8axvC C: citing same article (ref.) M: map data used to model this data |
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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: 53011.754 Da / Num. of mol.: 12 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 #2: Chemical | ChemComp-ZN / #3: Chemical | ChemComp-SAH / 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 covalently linked to m7GMP cap via H37 Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.72 MDa / Experimental value: YES | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Chikungunya virus strain S27-African prototype | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Trichoplusia ni (cabbage looper) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.6 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Sample incubated with 10x molar excess of m7GTP, SAH and 27mer CHIKV RNA to form the complex prior to freezing in the presence of magnesium. | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / 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
Microscopy | Model: TFS TALOS |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm |
Image recording | Average exposure time: 38 sec. / Electron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 773 |
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: 111668 | |||||||||||||||||||||||||||
Symmetry | Point symmetry: C12 (12 fold cyclic) | |||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 10758 / Symmetry type: POINT | |||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient | |||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6Z0V Accession code: 6Z0V / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||
Displacement parameters | Biso mean: 99.44 Å2 | |||||||||||||||||||||||||||
Refine LS restraints |
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