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- PDB-8acc: CryoEM structure of sweet potato mild mottle virus VLP -

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Basic information

Entry
Database: PDB / ID: 8acc
TitleCryoEM structure of sweet potato mild mottle virus VLP
Components
  • Polyprotein
  • Single-stranded RNA
KeywordsVIRUS LIKE PARTICLE / Plant virus / coat protein / VLP
Function / homology
Function and homology information


viral capsid / viral RNA genome replication / RNA-directed RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding
Similarity search - Function
Potyvirus coat protein / Potyvirus coat protein / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesSweet potato mild mottle virus
Nicotiana tabacum (common tobacco)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsJaved, A. / Byrne, B.M. / Ranson, N. / Lomonosoff, G.
Funding support Spain, United Kingdom, 4items
OrganizationGrant numberCountry
Ministerio de Ciencia e Innovacion (MCIN)PID2019-105692RB-100 Spain
Ministerio de Ciencia e Innovacion (MCIN)CEX2019-000902-S Spain
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R001669/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T004703/1 United Kingdom
CitationJournal: Commun Biol / Year: 2023
Title: CryoEM and stability analysis of virus-like particles of potyvirus and ipomovirus infecting a common host.
Authors: Ornela Chase / Abid Javed / Matthew J Byrne / Eva C Thuenemann / George P Lomonossoff / Neil A Ranson / Juan José López-Moya /
Abstract: Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, ...Sweet potato feathery mottle virus (SPFMV) and Sweet potato mild mottle virus (SPMMV) are members of the genera Potyvirus and Ipomovirus, family Potyviridae, sharing Ipomoea batatas as common host, but transmitted, respectively, by aphids and whiteflies. Virions of family members consist of flexuous rods with multiple copies of a single coat protein (CP) surrounding the RNA genome. Here we report the generation of virus-like particles (VLPs) by transient expression of the CPs of SPFMV and SPMMV in the presence of a replicating RNA in Nicotiana benthamiana. Analysis of the purified VLPs by cryo-electron microscopy, gave structures with resolutions of 2.6 and 3.0 Å, respectively, showing a similar left-handed helical arrangement of 8.8 CP subunits per turn with the C-terminus at the inner surface and a binding pocket for the encapsidated ssRNA. Despite their similar architecture, thermal stability studies reveal that SPMMV VLPs are more stable than those of SPFMV.
History
DepositionJul 5, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Data collection / Source and taxonomy / Category: chem_comp_atom / chem_comp_bond / entity_src_gen
Item: _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyprotein
B: Single-stranded RNA


Theoretical massNumber of molelcules
Total (without water)35,7692
Polymers35,7692
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polyprotein


Mass: 34282.840 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sweet potato mild mottle virus / Production host: Nicotiana benthamiana (plant) / References: UniProt: Q599X9
#2: RNA chain Single-stranded RNA


Mass: 1485.872 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Nicotiana tabacum (common tobacco)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Sweet potato mild mottle virus / Type: VIRUS / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 38 kDa/nm / Experimental value: NO
Source (natural)Organism: Sweet potato mild mottle virus
Source (recombinant)Organism: Nicotiana benthamiana (plant)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Ipomoea batatas
Virus shellDiameter: 130 nm
Buffer solutionpH: 7
Buffer componentConc.: 50 mM / Name: Sodium phosphate / Formula: NaPO4
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3ul of sample applied to each grid.

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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 defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2260
Image scansSampling size: 14 µm

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19model refinement
14Cootmodel refinement
15ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Helical symmertyAngular rotation/subunit: 41.29 ° / Axial rise/subunit: 3.97 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 329573
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38705 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 123.7 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 55.25 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00191825
ELECTRON MICROSCOPYf_angle_d0.47792513
ELECTRON MICROSCOPYf_chiral_restr0.0389303
ELECTRON MICROSCOPYf_plane_restr0.0023309
ELECTRON MICROSCOPYf_dihedral_angle_d6.2353304

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