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- EMDB-15578: CryoEM structure of the Chikungunya virus nsP1 capping pores in c... -

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Basic information

Entry
Database: EMDB / ID: EMD-15578
TitleCryoEM structure of the Chikungunya virus nsP1 capping pores in covalent complex with a 7GMP cap structure
Map data
Sample
  • Complex: CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37
    • Protein or peptide: Polyprotein P1234
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
Function / homology
Function and homology information


ADP-ribose 1''-phosphate phosphatase / host cell filopodium / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide adenylyltransferase / polynucleotide 5'-phosphatase activity / poly(A) RNA polymerase activity / mRNA modification / regulation of cytoskeleton organization ...ADP-ribose 1''-phosphate phosphatase / host cell filopodium / mRNA methyltransferase activity / mRNA 5'-triphosphate monophosphatase activity / mRNA 5'-phosphatase / polynucleotide adenylyltransferase / polynucleotide 5'-phosphatase activity / poly(A) RNA polymerase activity / mRNA modification / 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 / methylation / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / RNA helicase activity / symbiont-mediated suppression of host innate immune response / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / GTP binding / host cell nucleus / host cell plasma membrane / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / metal ion binding / membrane
Similarity search - Function
: / Alphavirus nsp2 protease (nsp2pro) domain / Alphavirus nsP2 protease domain superfamily / : / Peptidase family C9 / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsp2 protease (nsp2pro) domain profile. / : / Non-structural protein 3, zinc-binding domain / Viral methyltransferase ...: / Alphavirus nsp2 protease (nsp2pro) domain / Alphavirus nsP2 protease domain superfamily / : / Peptidase family C9 / Tomato mosaic virus helicase, N-terminal domain / Alphavirus nsp2 protease (nsp2pro) domain profile. / : / Non-structural protein 3, zinc-binding domain / 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 core domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein 3, X-domain-like / Appr-1"-p processing enzyme / Macro domain / Macro domain profile. / Macro domain / 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: 3.2 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 10, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateMay 3, 2023-
Current statusMay 3, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15578.png

Downloads & links

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Map

FileDownload / File: emd_15578.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 307.8 Å		0.86 Å/pix.
x 360 pix.
= 307.8 Å		0.86 Å/pix.
x 360 pix.
= 307.8 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0576
Minimum - Maximum-0.0018233435 - 1.8296849
Average (Standard dev.)0.003890528 (±0.048913736)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 307.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15578_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15578_half_map_2.map
Projections & Slices
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Sample components

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Entire : CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37

EntireName: CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37
Components
  • Complex: CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37
    • Protein or peptide: Polyprotein P1234
  • Ligand: ZINC ION
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE

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Supramolecule #1: CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37

SupramoleculeName: CHIKV nsP1 capping pores covalently linked to m7GMP cap via H37
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Chikungunya virus strain S27-African prototype
Molecular weightTheoretical: 720 KDa

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Macromolecule #1: Polyprotein P1234

MacromoleculeName: Polyprotein P1234 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Chikungunya virus strain S27-African prototype / Strain: S27-African prototype
Molecular weightTheoretical: 53.011754 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: MDPVYVDIDA DSAFLKALQR AYPMFEVEPR QVTPND(PJ3)ANA RAFSHLAIKL IEQEIDPDST ILDIGSAPAR RMMSDR KYH CVCPMRSAED PERLANYARK LASAAGKVLD RNISGKIGDL QAVMAVPDTE TPTFCLHTDV SCRQRADVAI YQDVYAV HA PTSLYHQAIK ...String:
MDPVYVDIDA DSAFLKALQR AYPMFEVEPR QVTPND(PJ3)ANA RAFSHLAIKL IEQEIDPDST ILDIGSAPAR RMMSDR KYH CVCPMRSAED PERLANYARK LASAAGKVLD RNISGKIGDL QAVMAVPDTE TPTFCLHTDV SCRQRADVAI YQDVYAV HA PTSLYHQAIK GVRLAYWVGF DTTPFMYNAM AGAYPSYSTN WADEQVLKAK NIGLCSTDLT EGRRGKLSIM RGKKLEPC D RVLFSVGSTL YPESRKLLKS WHLPSVFHLK GKLSFTCRCD TVVSCEGYVV KRITMSPGLY GKTTGYAVTH HADGFLMCK TTDTVDGERV SFSVCTYVPA TICDQMTGIL ATEVTPEDAQ KLLVGLNQRI VVNGRTQRNT NTMKNYMIPV VAQAFSKWAK ECRKDMEDE KLLGVRERTL TCCCLWAFKK QKTHTVYKRP DTQSIQKVQA EFDSFVVPSL WSSGLSIPLR TRIK

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 12 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: S-ADENOSYL-L-HOMOCYSTEINE

MacromoleculeName: S-ADENOSYL-L-HOMOCYSTEINE / type: ligand / ID: 3 / Number of copies: 12 / Formula: SAH
Molecular weightTheoretical: 384.411 Da
Chemical component information

ChemComp-SAH:
S-ADENOSYL-L-HOMOCYSTEINE

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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

Concentration0.2 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
25.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
150.0 mMNaClSodium chloride
2.0 mMTCEPTris(2-carboxyethyl)phosphine hydrochloride
0.06 %fos-choline12Dodecylphosphocholine
2.0 mMMgCl2Magnesium chloride
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsSample incubated with 10x molar excess of m7GTP, SAH and 27mer CHIKV RNA to form the complex prior to freezing in the presence of magnesium.

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Electron microscopy

MicroscopeTFS TALOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 773 / Average exposure time: 38.0 sec. / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm

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Image processing

Particle selectionNumber selected: 111668
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 10758
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-8apx:
CryoEM structure of the Chikungunya virus nsP1 capping pores in covalent complex with a 7GMP cap structure

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