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- PDB-6qoz: CryoEM reconstruction of Cowpea Mosaic Virus (CPMV) bound to an A... -

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Basic information

Entry
Database: PDB / ID: 6qoz
TitleCryoEM reconstruction of Cowpea Mosaic Virus (CPMV) bound to an Affimer reagent
Components
  • Affimer binding protein
  • Cowpea mosaic virus large subunit
  • RNA2 polyprotein
KeywordsVIRUS / CPMV / COMOVIRIDAE / PICORNAVIRALES / AFFIMEr REAGENT / antibody-like
Function / homology
Function and homology information


transport of virus in host, cell to cell / host cell plasmodesma / T=3 icosahedral viral capsid / host cell nucleus / GTP binding / structural molecule activity / DNA binding / RNA binding
Similarity search - Function
Large coat protein / RNA2 polyprotein / Large coat protein / Small coat protein / Jelly Rolls - #20 / Viral coat protein subunit / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesCowpea mosaic virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsHesketh, E.L. / Tiede, C. / Adamson, H. / Adams, T.L. / Byrne, M.J. / Meshcheriakova, Y. / Lomonossoff, G.P. / Kruse, I. / McPherson, M.J. / Tomlinson, D.C. / Ranson, N.A.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L021250/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/R00160X/1 United Kingdom
Wellcome Trust090932/Z/09/Z United Kingdom
Citation
Journal: Sci Rep / Year: 2019
Title: Affimer reagents as tools in diagnosing plant virus diseases.
Authors: Emma L Hesketh / Christian Tiede / Hope Adamson / Thomas L Adams / Matthew J Byrne / Yulia Meshcheriakova / Inga Kruse / Michael J McPherson / George P Lomonossoff / Darren C Tomlinson / Neil A Ranson /
Abstract: Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to ...Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests.
#1: Journal: Nat Commun / Year: 2015
Title: Mechanisms of assembly and genome packaging in an RNA virus revealed by high-resolution cryo-EM.
Authors: Emma L Hesketh / Yulia Meshcheriakova / Kyle C Dent / Pooja Saxena / Rebecca F Thompson / Joseph J Cockburn / George P Lomonossoff / Neil A Ranson /
Abstract: Cowpea mosaic virus is a plant-infecting member of the Picornavirales and is of major interest in the development of biotechnology applications. Despite the availability of >100 crystal structures of ...Cowpea mosaic virus is a plant-infecting member of the Picornavirales and is of major interest in the development of biotechnology applications. Despite the availability of >100 crystal structures of Picornavirales capsids, relatively little is known about the mechanisms of capsid assembly and genome encapsidation. Here we have determined cryo-electron microscopy reconstructions for the wild-type virus and an empty virus-like particle, to 3.4 Å and 3.0 Å resolution, respectively, and built de novo atomic models of their capsids. These new structures reveal the C-terminal region of the small coat protein subunit, which is essential for virus assembly and which was missing from previously determined crystal structures, as well as residues that bind to the viral genome. These observations allow us to develop a new model for genome encapsidation and capsid assembly.
History
DepositionFeb 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: RNA2 polyprotein
B: Cowpea mosaic virus large subunit
D: RNA2 polyprotein
E: Cowpea mosaic virus large subunit
F: RNA2 polyprotein
G: Cowpea mosaic virus large subunit
H: RNA2 polyprotein
I: Cowpea mosaic virus large subunit
C: Affimer binding protein


Theoretical massNumber of molelcules
Total (without water)256,5679
Polymers256,5679
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: ELISA
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area35970 Å2
ΔGint-197 kcal/mol
Surface area82100 Å2
MethodPISA

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Components

#1: Protein
RNA2 polyprotein / Genome polyprotein M / M RNA polyprotein / Middle component RNA polyprotein / P2


Mass: 20961.564 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cowpea mosaic virus / Strain: SB / Production host: Nicotiana benthamiana (plant) / References: UniProt: P03599
#2: Protein
Cowpea mosaic virus large subunit / / Genome polyprotein M / P2


Mass: 40858.434 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cowpea mosaic virus / Strain: SB / Production host: Nicotiana benthamiana (plant) / References: UniProt: P03599
#3: Protein Affimer binding protein


Mass: 9286.674 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Cowpea mosaic virusCOMPLEXall0MULTIPLE SOURCES
2Cowpea mosaic virusVIRUS#1-#21RECOMBINANT
3AffimerCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Cowpea mosaic virus12264
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Nicotiana benthamiana (plant)4100
23Escherichia coli (E. coli)562
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Cowpea mosaic virus
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281 K

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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 / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 45 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2980

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 144258
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15441 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
14N6U

4n6u

1
25A32

5a32

1

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