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- PDB-7ata: Nudaurelia capensis omega virus procapsid: virus-like particles e... -

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Basic information

Entry
Database: PDB / ID: 7ata
TitleNudaurelia capensis omega virus procapsid: virus-like particles expressed in Nicotiana benthamiana
Components(p70) x 2
KeywordsVIRUS LIKE PARTICLE / ICOSAHEDRAL VIRUS / AUTO-CATALYTIC CLEAVAGE / VIRUS MATURATION / TRANSIENT EXPRESSION
Function / homologyPeptidase N2 / Peptidase family A21 / Viral coat protein subunit / p70
Function and homology information
Biological speciesNudaurelia capensis omega virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.63 Å
AuthorsCastells-Graells, R. / Ribeiro, J.R.S. / Domitrovic, T. / Hesketh, E.L. / Scarff, C.A. / Johnson, J.E. / Ranson, N.A. / Lawson, D.M. / Lomonossoff, G.P.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BBS/E/J/000PR9794 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/L014130/1 United Kingdom
John Innes FoundationNone United Kingdom
CitationJournal: Commun Biol / Year: 2021
Title: Plant-expressed virus-like particles reveal the intricate maturation process of a eukaryotic virus.
Authors: Roger Castells-Graells / Jonas R S Ribeiro / Tatiana Domitrovic / Emma L Hesketh / Charlotte A Scarff / John E Johnson / Neil A Ranson / David M Lawson / George P Lomonossoff /
Abstract: Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus ...Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.
History
DepositionOct 29, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: p70
aa: p70
B: p70
bb: p70
C: p70
cc: p70
D: p70
dd: p70


Theoretical massNumber of molelcules
Total (without water)279,6408
Polymers279,6408
Non-polymers00
Water0
1
A: p70
aa: p70
B: p70
bb: p70
C: p70
cc: p70
D: p70
dd: p70
x 60


Theoretical massNumber of molelcules
Total (without water)16,778,383480
Polymers16,778,383480
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation59
Buried area17180 Å2
ΔGint-67 kcal/mol
Surface area95440 Å2

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Components

#1: Protein
p70


Mass: 62094.828 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nudaurelia capensis omega virus / Production host: Nicotiana benthamiana (plant) / Tissue (production host): leaf / References: UniProt: Q4TVS9
#2: Protein
p70


Mass: 7815.102 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nudaurelia capensis omega virus / Production host: Nicotiana benthamiana (plant) / Tissue (production host): leaf / References: UniProt: Q4TVS9

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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: Nudaurelia capensis omega virus / Type: VIRUS
Details: The codon-optimized sequence was transiently expressed in Nicotiana benthamiana
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 16.76 MDa / Experimental value: NO
Source (natural)Organism: Nudaurelia capensis omega virus
Source (recombinant)Organism: Nicotiana benthamiana (plant) / Cell: leaf tissue / Plasmid: pEAQ-HT
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Gonimbrasia cytherea
Virus shellName: coat / Diameter: 480 nm / Triangulation number (T number): 4
Buffer solutionpH: 7.6 / Details: NULL
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: NULL
Specimen supportGrid material: COPPER
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 72 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8554 / Details: NULL

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.14model fitting
8Coot0.9model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.18.2model refinement
Image processingDetails: NULL
CTF correctionDetails: NULL / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 6.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5426 / Symmetry type: POINT
Atomic model buildingB value: 105.8 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: The model was built and refined against a density modified map generated using phenix.resolve_cryo_em. This used the Relion half maps as inputs and improved the resolution to 6.6 Angstrom.
Atomic model buildingPDB-ID: 7ANM

7anm

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