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- PDB-9qvf: Turnip Crinkle Virus: virus-like particles (TCV-P38+1) -

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Basic information

Entry
Database: PDB / ID: 9qvf
TitleTurnip Crinkle Virus: virus-like particles (TCV-P38+1)
ComponentsCapsid protein
KeywordsVIRUS LIKE PARTICLE / ssRNA plant virus / icosahedral / RNA packaging
Function / homologyPlant viruses icosahedral capsid proteins 'S' region signature. / Icosahedral viral capsid protein, S domain / Viral coat protein (S domain) / T=3 icosahedral viral capsid / Viral coat protein subunit / symbiont-mediated suppression of host innate immune response / structural molecule activity / RNA binding / Capsid protein
Function and homology information
Biological speciesTurnip crinkle virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsSaunders, K. / Shah, S. / Peyret, H. / Meshcheriakova, Y. / Richardson, J. / Eltschkner, S. / Lawson, D.M. / Lomonossoff, G.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X01097X/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/Y005732/1 United Kingdom
CitationJournal: J Mol Biol / Year: 2025
Title: The Specificity of RNA Packaging in Isometric RNA Plant Viruses is Principally Determined by Replication.
Authors: Keith Saunders / Sachin N Shah / Hadrien Peyret / Yulia Meshcheriakova / Jake Richardson / Sandra Eltschkner / David M Lawson / George P Lomonossoff /
Abstract: A potato virus X (PVX)-based transient expression system (pEff) that produces replicating RNA has been used to examine the specificity of RNA packaging in the isometric viruses, turnip crinkle virus ...A potato virus X (PVX)-based transient expression system (pEff) that produces replicating RNA has been used to examine the specificity of RNA packaging in the isometric viruses, turnip crinkle virus (TCV) and satellite tobacco necrosis virus-1 (STNV-1). Expression of the coat proteins from the subgenomic RNA derived from the replicating PVX genome results in the efficient production of virus-like particles (VLPs), indistinguishable in structure from native virus particles, and encapsidation of both the subgenomic RNA and truncated versions of the replicating genomic RNA. Non-specific encapsidation of host RNA (which is not replicating) could not be detected in this system, implying that replication is the major determinant of packaging in isometric as well as filamentous positive-strand RNA plant viruses. We further utilised the system to investigate the role of putative packaging signals previously identified within the coat protein open reading frames of both TCV and STNV-1. The results show that eliminating the hairpin structures previously identified as packaging signals has no detectable effect on the specificity of RNA packaging. Replacement of the 213 nucleotide sequence within the TCV coat protein coding region, believed to be important for genomic packaging, with an equivalent sequence codon-optimised for Plasmodium falciparum resulted in less efficient capsid formation and RNA packaging, but did not alter packaging specificity; addition of copies of the wild-type sequence did not complement the defects. We propose that replication is the major determinant of genome packaging specificity in plant RNA viruses, while packaging signals may play a role in packaging efficiency.
History
DepositionApr 11, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Aug 6, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein


Theoretical massNumber of molelcules
Total (without water)114,5133
Polymers114,5133
Non-polymers00
Water00
1
A: Capsid protein
B: Capsid protein
C: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)6,870,769180
Polymers6,870,769180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Capsid protein / Coat protein / p38


Mass: 38170.941 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Turnip crinkle virus / Strain: M / Gene: ORF4 / Production host: Nicotiana benthamiana (plant) / References: UniProt: P06663
Has protein modificationN

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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: Turnip crinkle virus / Type: VIRUS
Details: In the manuscript, this sample is designated TCV-P38+1. The complete P38 coat protein gene was inserted into the pEff vector together with an additional duplicated portion of P38 (hence +1). ...Details: In the manuscript, this sample is designated TCV-P38+1. The complete P38 coat protein gene was inserted into the pEff vector together with an additional duplicated portion of P38 (hence +1). Expression was initiated by infiltrating leaves of Nicotiana benthamiana with suspensions of Agrobacterium tumefaciens strain LBA4404 harbouring this vector.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 6.86 MDa / Experimental value: NO
Source (natural)Organism: Turnip crinkle virus / Strain: M
Source (recombinant)Organism: Nicotiana benthamiana (plant)
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Brassica rapa subsp. rapa
Virus shellName: capsid / Diameter: 340 nm / Triangulation number (T number): 3
Buffer solutionpH: 7.4 / Details: 1 mM MgSO4 1mM NaPO4 and pH7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: glow discharged for 60 seconds at 8 mA using an ACE 200 (Leica Microsystems)
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: TFS TALOS F200C
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN ELSA 698 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 3.49 sec. / Electron dose: 30 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6269
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5.3particle selection
2EPU3.7.1.6940image acquisition
4cryoSPARC4.5.3CTF correction
7UCSF ChimeraX1.8model fitting
8Coot0.9.8.95 EL (ccp4)model fitting
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
14PHENIX1.21.2_5419:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 92873
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48900 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 65.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingPDB-ID: 3ZX8

3zx8


Accession code: 3ZX8 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00378120
ELECTRON MICROSCOPYf_angle_d0.436106092
ELECTRON MICROSCOPYf_dihedral_angle_d3.78810816
ELECTRON MICROSCOPYf_chiral_restr0.04111808
ELECTRON MICROSCOPYf_plane_restr0.00313848

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