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- PDB-8aow: CryoEM structure of the Chikungunya virus nsP1 capping pores in c... -

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Basic information

Entry
Database: PDB / ID: 8aow
TitleCryoEM structure of the Chikungunya virus nsP1 capping pores in complex with m7GTP and SAH ligands
ComponentsmRNA-capping enzyme nsP1
KeywordsVIRAL 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
: / : / : / Non-structural protein 3, zinc-binding domain / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsP2 protease domain superfamily / Alphavirus nsp2 protease (nsp2pro) domain / Peptidase family C9 / Alphavirus nsp2 protease (nsp2pro) domain profile. / Viral methyltransferase ...: / : / : / Non-structural protein 3, zinc-binding domain / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsP2 protease domain superfamily / Alphavirus nsp2 protease (nsp2pro) domain / Peptidase family C9 / Alphavirus nsp2 protease (nsp2pro) domain profile. / Viral methyltransferase / Alphavirus-like methyltransferase (MT) domain / Alphavirus-like methyltransferase (MT) domain profile. / Tymovirus, RNA-dependent RNA polymerase / RNA dependent RNA polymerase / Viral (Superfamily 1) RNA helicase / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein 3, X-domain-like / Macro domain / Appr-1"-p processing enzyme / Macro domain / Macro domain profile. / Macro domain-like / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
7-METHYL-GUANOSINE-5'-TRIPHOSPHATE / S-ADENOSYL-L-HOMOCYSTEINE / Polyprotein P1234
Similarity search - Component
Biological speciesChikungunya virus strain S27-African prototype
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsJones, R. / Hons, M. / Reguera, J.
Funding support France, 1items
OrganizationGrant numberCountry
ATIP-Avenir France
CitationJournal: 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
DepositionAug 8, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: mRNA-capping enzyme nsP1
C: mRNA-capping enzyme nsP1
E: mRNA-capping enzyme nsP1
G: mRNA-capping enzyme nsP1
I: mRNA-capping enzyme nsP1
K: mRNA-capping enzyme nsP1
M: mRNA-capping enzyme nsP1
O: mRNA-capping enzyme nsP1
Q: mRNA-capping enzyme nsP1
S: mRNA-capping enzyme nsP1
V: mRNA-capping enzyme nsP1
X: mRNA-capping enzyme nsP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)743,79772
Polymers731,35812
Non-polymers12,44060
Water5,837324
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 12 molecules ACEGIKMOQSVX

#1: Protein
mRNA-capping enzyme nsP1 / Non-structural protein 1


Mass: 60946.473 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, Transferases; Transferring one-carbon groups; Methyltransferases, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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Non-polymers , 5 types, 384 molecules

#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn
#3: Chemical
ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-MGP / 7-METHYL-GUANOSINE-5'-TRIPHOSPHATE


Mass: 538.215 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C11H19N5O14P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 324 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CHIKV nsP1 capping pores in complex with m7GTP and SAH ligand with magnesium cofactor.
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.72 MDa / Experimental value: YES
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
2150 mMSodium chlorideNaClSodium chloride1
32 mMTris(2-carboxyethyl)phosphine hydrochlorideTCEP1
40.06 %Dodecylphosphocholinefos-choline121
52 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Specimen was incubated for 2 hours with 0.5mM SAH and m7GTP prior to plunge freezing
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 70 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.85 sec. / Electron dose: 38 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3077
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 338748
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 253384 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6Z0V

6z0v

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 41.64 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007344244
ELECTRON MICROSCOPYf_angle_d0.758660084
ELECTRON MICROSCOPYf_chiral_restr0.04776792
ELECTRON MICROSCOPYf_plane_restr0.00667536
ELECTRON MICROSCOPYf_dihedral_angle_d7.40716360

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