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- PDB-8opk: Local refinement of cubic assembly from truncated PVY coat protei... -

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Basic information

Entry
Database: PDB / ID: 8opk
TitleLocal refinement of cubic assembly from truncated PVY coat protein with K176C mutation
ComponentsGenome polyprotein
KeywordsVIRUS LIKE PARTICLE / cubes / trCP / K176C / Potyvirus / PVY
Function / homologyPotyvirus coat protein / Potyvirus coat protein / viral capsid / Genome polyprotein
Function and homology information
Biological speciesPotato virus Y strain NTN
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsKavcic, L. / Kezar, A. / Podobnik, M.
Funding support Slovenia, Czech Republic, 3items
OrganizationGrant numberCountry
Slovenian Research AgencyP1-0391 Slovenia
Slovenian Research AgencyJ7-7248 Slovenia
Ministry of Education, Youth and Sports of the Czech RepublicLM2018127 Czech Republic
CitationJournal: Commun Chem / Year: 2024
Title: From structural polymorphism to structural metamorphosis of the coat protein of flexuous filamentous potato virus Y.
Authors: Luka Kavčič / Andreja Kežar / Neža Koritnik / Magda Tušek Žnidarič / Tajda Klobučar / Žiga Vičič / Franci Merzel / Ellie Holden / Justin L P Benesch / Marjetka Podobnik /
Abstract: The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid ...The structural diversity and tunability of the capsid proteins (CPs) of various icosahedral and rod-shaped viruses have been well studied and exploited in the development of smart hybrid nanoparticles. However, the potential of CPs of the wide-spread flexuous filamentous plant viruses remains to be explored. Here, we show that we can control the shape, size, RNA encapsidation ability, symmetry, stability and surface functionalization of nanoparticles through structure-based design of CP from potato virus Y (PVY). We provide high-resolution insight into CP-based self-assemblies, ranging from large polymorphic or monomorphic filaments to smaller annular, cubic or spherical particles. Furthermore, we show that we can prevent CP self-assembly in bacteria by fusion with a cleavable protein, enabling controlled nanoparticle formation in vitro. Understanding the remarkable structural diversity of PVY CP not only provides possibilities for the production of biodegradable nanoparticles, but may also advance future studies of CP's polymorphism in a biological context.
History
DepositionApr 7, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 24, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
Aa: Genome polyprotein
Ab: Genome polyprotein
Ac: Genome polyprotein
Ad: Genome polyprotein
Ae: Genome polyprotein
Af: Genome polyprotein
Ag: Genome polyprotein
Ah: Genome polyprotein
Ai: Genome polyprotein
Aj: Genome polyprotein
Ak: Genome polyprotein
Al: Genome polyprotein
Am: Genome polyprotein
An: Genome polyprotein
Ao: Genome polyprotein
Ap: Genome polyprotein


Theoretical massNumber of molelcules
Total (without water)357,76716
Polymers357,76716
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Genome polyprotein


Mass: 22360.424 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Potato virus Y strain NTN / Strain: NTN / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0A7DIW7

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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: truncated coat protein (dN49C40) with C-terminal His tag and K176C mutation
Type: COMPLEX / Details: structurally homogenous cubic particles / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.315 MDa / Experimental value: YES
Source (natural)Organism: Potato virus Y strain NTN
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) / Plasmid: pET28a
Buffer solutionpH: 7.4
Details: 1.8 mM KH2PO4, 10.1 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 300 nm
Image recordingAverage exposure time: 4 sec. / Electron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 9480
Image scansMovie frames/image: 32

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Processing

EM software
IDNameCategoryDetails
1cryoSPARCparticle selectionBlob picker
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstructionLocal refinement
CTF correctionDetails: patchCTF correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1580128
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 843116
Details: Final cryoEM map was obtained by local refinement from C4 symmetry expanded particles.
Symmetry type: POINT

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