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- EMDB-17056: Stacked-ring assembly from truncated PVY coat protein with L99C m... -

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Basic information

Entry
Database: EMDB / ID: EMD-17056
TitleStacked-ring assembly from truncated PVY coat protein with L99C mutation
Map datatrCPL99C stacked double ring filament sharp symmetric cryoEM map (C8)
Sample
  • Complex: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
    • Protein or peptide: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
Keywordsstacked-ring / trCP / L99C / Potyvirus / PVY / VIRUS LIKE PARTICLE
Biological speciesPotato virus Y strain NTN
Methodhelical reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsKavcic L / Kezar A / Podobnik M
Funding support Slovenia, 2 items
OrganizationGrant numberCountry
Slovenian Research AgencyP1-0391 Slovenia
Slovenian Research AgencyJ7-7248 Slovenia
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, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17056.png

Downloads & links

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Map

FileDownload / File: emd_17056.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationtrCPL99C stacked double ring filament sharp symmetric cryoEM map (C8)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 300 pix.
= 290.4 Å		0.97 Å/pix.
x 300 pix.
= 290.4 Å		0.97 Å/pix.
x 300 pix.
= 290.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.968 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.472
Minimum - Maximum-1.0208172 - 2.623802
Average (Standard dev.)0.031508278 (±0.15663081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 290.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17056_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: trCPL99C stacked double ring filament raw cryoEM map

Fileemd_17056_additional_1.map
AnnotationtrCPL99C stacked double ring filament raw cryoEM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: trCPL99C stacked double ring filament half B cryoEM map

Fileemd_17056_half_map_1.map
AnnotationtrCPL99C stacked double ring filament half B cryoEM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: trCPL99C stacked double ring filament half A cryoEM map

Fileemd_17056_half_map_2.map
AnnotationtrCPL99C stacked double ring filament half A cryoEM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : truncated coat protein (dN49C40) with C-terminal His tag and L99C...

EntireName: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
Components
  • Complex: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
    • Protein or peptide: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation

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Supramolecule #1: truncated coat protein (dN49C40) with C-terminal His tag and L99C...

SupramoleculeName: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The sample contains 2 types of filaments (helical and with double ring in a head-to-tail arrangement as the repeating unit).
Source (natural)Organism: Potato virus Y strain NTN

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Macromolecule #1: truncated coat protein (dN49C40) with C-terminal His tag and L99C...

MacromoleculeName: truncated coat protein (dN49C40) with C-terminal His tag and L99C mutation
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Potato virus Y strain NTN / Strain: NTN
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GITSKMRMPK SKGATVLNLE HLLEYAPQQI DISNTRATQS QFDTWYEAVQ CAYDIGETEM PTVMNGLMVW CIENGTSPNI NGVWVMMDGD EQVEYPLKPI VENAKPTLRQ IMAHFSDVAE AYIEMRNKKE PYMPRYGLVR NLRDGSLARY AFDFYEVTSR TPVRAREAHI ...String:
GITSKMRMPK SKGATVLNLE HLLEYAPQQI DISNTRATQS QFDTWYEAVQ CAYDIGETEM PTVMNGLMVW CIENGTSPNI NGVWVMMDGD EQVEYPLKPI VENAKPTLRQ IMAHFSDVAE AYIEMRNKKE PYMPRYGLVR NLRDGSLARY AFDFYEVTSR TPVRAREAHI QMKAAALKSE NLYFQGLEHH HHHH

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.4
Details: 1.8 mM KH2PO4, 10.1 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4
VitrificationCryogen name: ETHANE
DetailsThe sample was polymorphic.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 1398 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000

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Image processing

DetailsImage processing steps were done both with RELION and cryoSPARC.
Final reconstructionApplied symmetry - Helical parameters - Δz: 80.13 Å
Applied symmetry - Helical parameters - Δ&Phi: -8.21 °
Applied symmetry - Helical parameters - Axial symmetry: C8 (8 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 17349
Segment selectionNumber selected: 193175 / Software - Name: crYOLO / Details: CrYOLO picking using a pre-trained model
Startup modelType of model: NONE
Details: Initial model was obtained by ab-initio reconstruction
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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