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- EMDB-15704: Structure of an open form of CHIKV nsP1 capping pores -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-15704
TitleStructure of an open form of CHIKV nsP1 capping pores
Map data
Sample
  • Complex: Chikungunya nsP1 in an open form
    • Protein or peptide: mRNA-capping enzyme nsP1
  • Ligand: ZINC ION
  • Ligand: 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one
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
: / : / : / 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
AuthorsReguera J / Jones R / Hons M
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
DepositionSep 1, 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_15704.png

Downloads & links

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Map

FileDownload / File: emd_15704.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 500 pix.
= 419.5 Å		0.84 Å/pix.
x 500 pix.
= 419.5 Å		0.84 Å/pix.
x 500 pix.
= 419.5 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.839 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-2.0879378 - 3.3930538
Average (Standard dev.)0.00039296487 (±0.058194607)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 419.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15704_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15704_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Chikungunya nsP1 in an open form

EntireName: Chikungunya nsP1 in an open form
Components
  • Complex: Chikungunya nsP1 in an open form
    • Protein or peptide: mRNA-capping enzyme nsP1
  • Ligand: ZINC ION
  • Ligand: 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one

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Supramolecule #1: Chikungunya nsP1 in an open form

SupramoleculeName: Chikungunya nsP1 in an open form / 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: mRNA-capping enzyme nsP1

MacromoleculeName: mRNA-capping enzyme nsP1 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / 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: 12 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one

MacromoleculeName: 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one / type: ligand / ID: 3 / Number of copies: 12 / Formula: MY6
Molecular weightTheoretical: 165.153 Da
Chemical component information

ChemComp-MY6:
2-amino-7-methyl-1,7-dihydro-6H-purin-6-one / 7-Methylguanine

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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

Concentration0.15 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMNaClSodium chlorideSodium chloride
1.0 mMtris(2-carboxyethyl)phosphineTCEP
0.005 %glyco-diosgeninGDN
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: 98 % / Chamber temperature: 25 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was incubated with an RNA with a 5' methyl guanosine cap prior to vitrification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 44.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6zou

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 167543

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Atomic model buiding 1

Initial modelPDB ID:

6zou

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8axv:
Structure of an open form of CHIKV nsP1 capping pores

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