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- PDB-7y38: Molecular architecture of the chikungunya virus replication complex -

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Basic information

Entry
Database: PDB / ID: 7y38
TitleMolecular architecture of the chikungunya virus replication complex
Components
  • Protease nsP2
  • RNA (5'-R(P*CP*CP*A)-3')
  • RNA-directed RNA polymerase nsP4
  • mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
KeywordsVIRAL PROTEIN/RNA / replicase / replication complex / viral RNA replication / capping / helicase / protease / RNA-dependent RNA polymerase / RdRp / VIRAL PROTEIN / VIRAL PROTEIN-RNA complex
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
ADENOSINE-5'-TRIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / RNA / Polyprotein P1234
Similarity search - Component
Biological speciesChikungunya virus strain S27-African prototype
synthetic construct (others)
Onyong-nyong virus
in vitro transcription vector pT7-TP
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsTan, Y.B. / Luo, D.
Funding support Singapore, 1items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)T2EP30220-0020, 2021-T1-002-021 Singapore
CitationJournal: Sci Adv / Year: 2022
Title: Molecular architecture of the Chikungunya virus replication complex.
Authors: Yaw Bia Tan / David Chmielewski / Michelle Cheok Yien Law / Kuo Zhang / Yu He / Muyuan Chen / Jing Jin / Dahai Luo /
Abstract: To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we ...To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses.
History
DepositionJun 10, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
B: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
C: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
D: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
E: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
F: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
G: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
H: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
I: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
J: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
K: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
L: mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG)
X: RNA-directed RNA polymerase nsP4
Y: Protease nsP2
Z: RNA (5'-R(P*CP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)943,96451
Polymers930,81515
Non-polymers13,14936
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, cryoEM shows the density of nsP1, nsP2 and nsP4
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 14 molecules ABCDEFGHIJKLXY

#1: Protein
mRNA-capping enzyme nsP1,affinity-tag (strepII-3XFLAG) / Non-structural protein 1


Mass: 64328.938 Da / Num. of mol.: 12 / Mutation: H37A
Source method: isolated from a genetically manipulated source
Details: 1. affinity-tag (strepII-3XFLAG) insertion in between residue number 517-552; 2. replaced the last residue A535 with short peptide-tag NGL (corresponding to position 571-573)
Source: (gene. exp.) Chikungunya virus strain S27-African prototype, (gene. exp.) synthetic construct (others)
Strain: S27-African prototype / Plasmid: pCMV / Cell line (production host): Expi293 / Production host: Homo sapiens (human)
References: UniProt: Q8JUX6, Transferases; Transferring one-carbon groups; Methyltransferases, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein RNA-directed RNA polymerase nsP4


Mass: 68373.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Sourced from virus isolate cDNA, not available in uniprot database at the time of biocuration
Source: (gene. exp.) Onyong-nyong virus / Strain: Chad / Gene: nsP4 / Plasmid: pSUMO-LIC / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta II / Variant (production host): T1R
References: polynucleotide adenylyltransferase, RNA-directed RNA polymerase
#3: Protein Protease nsP2 / Non-structural protein 2 / nsP2


Mass: 89599.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: helicase region is built on visible 3D electron density map but not protease region
Source: (gene. exp.) Chikungunya virus strain S27-African prototype
Plasmid: pSUMO-LIC / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta II / Variant (production host): T1R
References: UniProt: Q8JUX6, polynucleotide 5'-phosphatase, Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases, nucleoside-triphosphate phosphatase, RNA helicase

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RNA chain , 1 types, 1 molecules Z

#4: RNA chain RNA (5'-R(P*CP*CP*A)-3')


Mass: 894.612 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) in vitro transcription vector pT7-TP(deltai) (others)

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Non-polymers , 3 types, 36 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Alphavirus Replication ComplexCOMPLEXAssembled replication complex composed of all full-length viral protein - nsP1, nsP2 and nsP4 bound with RNA and NTP#1-#40MULTIPLE SOURCES
2nsP1 dodecameric ringCOMPLEX12-mer ring#11RECOMBINANT
3nsP4 RdRp; docked within nsP1 central cavityCOMPLEXviral RdRp protein, single copy nsP4 slotted in the central pore of nsP1#21RECOMBINANT
4nsP2 helicase-protease; docked on top of viral complex with synthetic RNA boundCOMPLEXonly helicase density was observed#3-#41MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.877 MDaNO
210.72 MDaNO
310.1 MDaNO
410.1 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Chikungunya virus strain S27-African prototype371094
23Onyong-nyong virus2169701
34Chikungunya virus strain S27-African prototype371094
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
12Homo sapiens (human)9606Expi293pCMV
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 8
Details: 25mM HEPES, 75mM NaCl, 20mM KCl, 5mM MgOAc, 2.5mM MnCl2, 5mM TCEP, 1.25% Sucrose, pH8
Buffer component
IDConc.NameFormulaBuffer-ID
10.025 MolarHEPESHEPES1
20.075 MolarSodium ChlorideNaCl1
30.020 MolarPotassium ChlorideKCl1
40.005 MolarMagnesium AcetateMgOAc1
50.0025 MolarMangenese ChlorideMnCl21
60.005 MolarTCEPTCEP1
71.25 %SucroseSucrose1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was in vitro reconstituted and purified via affinity pulldown and ion-exchanged.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 273.15 K / Details: wait 45s, blot 2s at force -2

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1600 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 / Temperature (max): 80 K / Temperature (min): 70 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 34 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7865 / Details: movie-mode at 40 FPS
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selectiontemplate-based picker
2Topaz2.5.0particle selectionneural-network trained picker
3EPU2.14image acquisitiondata collection
5cryoSPARC3.3.1CTF correctionpatch CTF
8UCSF ChimeraX1.3model fittingfitting
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification3D Classification
13cryoSPARC3.3.13D reconstructionHomogenous/Heterogenous Refinement
14PHENIX1.2model refinementrefinement
15Coot0.9.5model refinementbuilding
Image processingDetails: The images were recorded in MRC non-gain normalized format.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2261181
Details: template-based picking and neural-network trained TOPAZ picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132510 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 63.66 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: ChimeraX was used to fit the 7DOP ring and built/refined using coot/phenix.
Atomic model buildingPDB-ID: 7DOP

7dop


Accession code: 7DOP / Pdb chain residue range: 1-516 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 112.09 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00254296
ELECTRON MICROSCOPYf_angle_d0.50773858
ELECTRON MICROSCOPYf_chiral_restr0.04068366
ELECTRON MICROSCOPYf_plane_restr0.00489280
ELECTRON MICROSCOPYf_dihedral_angle_d5.40937461

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