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- PDB-6z0v: CryoEM structure of the Chikungunya virus nsP1 complex -

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Basic information

Entry
Database: PDB / ID: 6z0v
TitleCryoEM structure of the Chikungunya virus nsP1 complex
ComponentsPolyprotein P1234
KeywordsVIRAL PROTEIN / Capping enzyme / monotopic membrane complex / membrane bending / membrane pore / replication complex / scaffold / Spherule formation / viral factory.
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
Biological speciesChikungunya virus strain S27-African prototype
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsReguera, J. / Jones, R. / Arranz-Avila, R.
Funding support France, 1items
OrganizationGrant numberCountry
ATIP-Avenir France
CitationJournal: Nature / Year: 2021
Title: Capping pores of alphavirus nsP1 gate membranous viral replication factories.
Authors: Rhian Jones / Gabriel Bragagnolo / Rocío Arranz / Juan Reguera /
Abstract: Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells. ...Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells. The remodelling of the host-cell membranes for the formation of these organelles is coupled to the membrane association of viral replication complexes and to RNA synthesis. These viral niches allow for the concentration of metabolites and proteins for the synthesis of viral RNA, and prevent the detection of this RNA by the cellular innate immune system. Here we present the cryo-electron microscopy structure of non-structural protein 1 (nsP1) of the alphavirus chikungunya virus, which is responsible for RNA capping and membrane binding of the viral replication machinery. The structure shows the enzyme in its active form, assembled in a monotopic membrane-associated dodecameric ring. The structure reveals the structural basis of the coupling between membrane binding, oligomerization and allosteric activation of the capping enzyme. The stoichiometry-with 12 active sites in a single complex-redefines viral replication complexes as RNA synthesis reactors. The ring shape of the complex implies it has a role in controlling access to the viral organelle and ensuring the exit of properly capped viral RNA. Our results provide high-resolution information about the membrane association of the replication machinery of positive-sense single-stranded RNA viruses, and open up avenues for the further characterization of viral replication on cell membranes and the generation of antiviral agents.
History
DepositionMay 11, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 21, 2020Group: Database references / Category: citation / Item: _citation.title
Revision 1.2Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Feb 10, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Polyprotein P1234
C: Polyprotein P1234
E: Polyprotein P1234
G: Polyprotein P1234
I: Polyprotein P1234
K: Polyprotein P1234
M: Polyprotein P1234
O: Polyprotein P1234
Q: Polyprotein P1234
S: Polyprotein P1234
V: Polyprotein P1234
X: Polyprotein P1234
hetero molecules


Theoretical massNumber of molelcules
Total (without water)637,55324
Polymers636,76812
Non-polymers78512
Water16,862936
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area81550 Å2
ΔGint-335 kcal/mol
Surface area194770 Å2
MethodPISA

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Components

#1: Protein
Polyprotein P1234 / P1234 / Non-structural polyprotein


Mass: 53064.031 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chikungunya virus 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, polynucleotide 5'- ...References: UniProt: Q8JUX6, Transferases; Transferring one-carbon groups; Methyltransferases, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, polynucleotide 5'-phosphatase, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, nucleoside-triphosphate phosphatase, RNA helicase, ADP-ribose 1''-phosphate phosphatase, polynucleotide adenylyltransferase, RNA-directed RNA polymerase
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 936 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: Chikungunya NsP1 capping pore complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.72 MDa / Experimental value: NO
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Source (recombinant)Organism: Baculovirus expression vector pFastBac1-HM
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
135 mMTris(hydroxymethyl)amino methane(HOCH2)3CNH21
2200 mMSodium chlorideNaClSodium chloride1
32 mMTCEPC9H15O6P1
40.03 % w/vfos-choline12C17H38NO4P1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE-PROPANE
Details: 3ul of sample was spotted onto the grid and hand blotted prior to plunge freezing.

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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 magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 38.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5148
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
2EPU1.5.0.32image acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9.2model fitting
9PHENIX1.17.1model refinement
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
CTF correctionDetails: CTF was estimated from movie frames aligned without dose-weighting. Per particle CTF correction was also performed with polished particles.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 263415
Details: Autopicking following generation of templates from 2D class averages
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94018 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: CC
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 35.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009642984
ELECTRON MICROSCOPYf_angle_d0.852658428
ELECTRON MICROSCOPYf_chiral_restr0.06066696
ELECTRON MICROSCOPYf_plane_restr0.0067416
ELECTRON MICROSCOPYf_dihedral_angle_d8.035635484

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