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- EMDB-15555: CryoEM structure of the Chikungunya virus nsP1 capping pores in c... -

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Basic information

Entry
Database: EMDB / ID: EMD-15555
TitleCryoEM structure of the Chikungunya virus nsP1 capping pores in complex with SAM
Map dataCHIKV nsP1 capping pores in complex with SAM ligand
Sample
  • Complex: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (SAM) ligand.
    • Protein or peptide: mRNA-capping enzyme nsP1
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYLMETHIONINES-Adenosyl methionine
  • Ligand: water
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
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJones R / Hons M / Reguera J
Funding support France, 1 items
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, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15555.png

Downloads & links

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Map

FileDownload / File: emd_15555.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCHIKV nsP1 capping pores in complex with SAM ligand
Voxel sizeX=Y=Z: 0.827 Å
Density
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.0917321 - 0.11743893
Average (Standard dev.)0.0008578563 (±0.0073250155)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: halfmap1

Fileemd_15555_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_15555_half_map_2.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CHIKV nsP1 capping pores in complex with S-adenosyl methionine (S...

EntireName: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (SAM) ligand.
Components
  • Complex: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (SAM) ligand.
    • Protein or peptide: mRNA-capping enzyme nsP1
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYLMETHIONINES-Adenosyl methionine
  • Ligand: water

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Supramolecule #1: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (S...

SupramoleculeName: CHIKV nsP1 capping pores in complex with S-adenosyl methionine (SAM) ligand.
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: mRNA-capping enzyme nsP1

MacromoleculeName: mRNA-capping enzyme nsP1 / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
EC number: Transferases; Transferring one-carbon groups; Methyltransferases
Source (natural)Organism: Chikungunya virus strain S27-African prototype / Strain: S27-African prototype
Molecular weightTheoretical: 59.975422 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MDPVYVDIDA DSAFLKALQR AYPMFEVEPR QVTPNDHANA RAFSHLAIKL IEQEIDPDST ILDIGSAPAR RMMSDRKYHC VCPMRSAED PERLANYARK LASAAGKVLD RNISGKIGDL QAVMAVPDTE TPTFCLHTDV SCRQRADVAI YQDVYAVHAP T SLYHQAIK ...String:
MDPVYVDIDA DSAFLKALQR AYPMFEVEPR QVTPNDHANA RAFSHLAIKL IEQEIDPDST ILDIGSAPAR RMMSDRKYHC VCPMRSAED PERLANYARK LASAAGKVLD RNISGKIGDL QAVMAVPDTE TPTFCLHTDV SCRQRADVAI YQDVYAVHAP T SLYHQAIK GVRLAYWVGF DTTPFMYNAM AGAYPSYSTN WADEQVLKAK NIGLCSTDLT EGRRGKLSIM RGKKLEPCDR VL FSVGSTL YPESRKLLKS WHLPSVFHLK GKLSFTCRCD TVVSCEGYVV KRITMSPGLY GKTTGYAVTH HADGFLMCKT TDT VDGERV SFSVCTYVPA TICDQMTGIL ATEVTPEDAQ KLLVGLNQRI VVNGRTQRNT NTMKNYMIPV VAQAFSKWAK ECRK DMEDE KLLGVRERTL TCCCLWAFKK QKTHTVYKRP DTQSIQKVQA EFDSFVVPSL WSSGLSIPLR TRIKWLLSKV PKTDL TPYS GDAQEARDAE KEAEEEREAE LTLEALPPLQ AAQEDVQVEI DVEQLEDRAG A

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 24 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: S-ADENOSYLMETHIONINE

MacromoleculeName: S-ADENOSYLMETHIONINE / type: ligand / ID: 3 / Number of copies: 24 / Formula: SAM
Molecular weightTheoretical: 398.437 Da
Chemical component information

ChemComp-SAM:
S-ADENOSYLMETHIONINE / S-Adenosyl methionine

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 4848 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
25.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
150.0 mMNaClSodium chlorideSodium chloride
2.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
0.06 %fos-choline12Dodecylphosphocholine
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsSpecimen was incubated with 0.5mM SAM

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4500 / Average exposure time: 4.0 sec. / Average electron dose: 42.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 366156
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D12 (2x12 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 69057
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6z0v

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-8aox:
CryoEM structure of the Chikungunya virus nsP1 capping pores in complex with SAM

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