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- PDB-3zx8: Cryo-EM reconstruction of native and expanded Turnip Crinkle virus -

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Basic information

Entry
Database: PDB / ID: 3zx8
TitleCryo-EM reconstruction of native and expanded Turnip Crinkle virus
ComponentsCAPSID PROTEIN
KeywordsVIRUS / GENOMIC RNA STRUCTURE / GENOME UNCOATING / SSRNA VIRUS / ICOSAHEDRAL
Function / homology
Function and homology information


T=3 icosahedral viral capsid / symbiont-mediated suppression of host innate immune response / virus-mediated perturbation of host defense response / structural molecule activity / RNA binding
Similarity search - Function
Plant viruses icosahedral capsid proteins 'S' region signature. / Icosahedral viral capsid protein, S domain / Viral coat protein (S domain) / Viral coat protein subunit
Similarity search - Domain/homology
Biological speciesTURNIP CRINKLE VIRUS
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 11.5 Å
AuthorsBakker, S.E. / Robottom, J. / Hogle, J.M. / Maeda, A. / Pearson, A.R. / Stockley, P.G. / Ranson, N.A. / Harrison, S.C.
Citation
Journal: J Mol Biol / Year: 2012
Title: Isolation of an asymmetric RNA uncoating intermediate for a single-stranded RNA plant virus.
Authors: Saskia E Bakker / Robert J Ford / Amy M Barker / Janice Robottom / Keith Saunders / Arwen R Pearson / Neil A Ranson / Peter G Stockley /
Abstract: We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the ...We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.
#1: Journal: J Mol Biol / Year: 1986
Title: Structure and assembly of turnip crinkle virus. I. X-ray crystallographic structure analysis at 3.2 A resolution.
Abstract: The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic ...The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.
History
DepositionAug 8, 2011Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 18, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 3, 2018Group: Data collection
Category: diffrn_radiation / diffrn_radiation_wavelength / em_software
Item: _em_software.image_processing_id
Revision 1.2May 8, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type
Remark 650 HELIX DETERMINATION METHOD: AUTHOR PROVIDED.
Remark 700 SHEET DETERMINATION METHOD: AUTHOR PROVIDED.

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
  • Imaged by Jmol
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
  • Imaged by Jmol
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-1863
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  • Superimposition on EM map
  • EMDB-1863
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Structure viewerMolecule:
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Assembly

Deposited unit
A: CAPSID PROTEIN
B: CAPSID PROTEIN
C: CAPSID PROTEIN


Theoretical massNumber of molelcules
Total (without water)113,2673
Polymers113,2673
Non-polymers00
Water00
1
A: CAPSID PROTEIN
B: CAPSID PROTEIN
C: CAPSID PROTEIN
x 60


Theoretical massNumber of molelcules
Total (without water)6,796,005180
Polymers6,796,005180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: CAPSID PROTEIN
B: CAPSID PROTEIN
C: CAPSID PROTEIN
x 5


  • icosahedral pentamer
  • 566 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)566,33415
Polymers566,33415
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: CAPSID PROTEIN
B: CAPSID PROTEIN
C: CAPSID PROTEIN
x 6


  • icosahedral 23 hexamer
  • 680 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)679,60118
Polymers679,60118
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein CAPSID PROTEIN / COAT PROTEIN / P38


Mass: 37755.586 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) TURNIP CRINKLE VIRUS / Strain: M / References: UniProt: P06663
Sequence detailsIN CHAIN C RESIDUES 1-52 DISORDERED AND NOT OBSERVED. IN CHAINS A AND B RESIDUES 1-80 DISORDERED ...IN CHAIN C RESIDUES 1-52 DISORDERED AND NOT OBSERVED. IN CHAINS A AND B RESIDUES 1-80 DISORDERED AND NOT OBSERVED.

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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: TURNIP CRINKLE VIRUS / Type: VIRUS
Buffer solutionName: 10 MM SODIUM PHOSPHATE PH 7.4, 10 MM MAGNESIUM SULPHATE
pH: 7.4
Details: 10 MM SODIUM PHOSPHATE PH 7.4, 10 MM MAGNESIUM SULPHATE
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, TEMPERATURE- 77, INSTRUMENT- DOUBLE SIDED AUTOMATED BLOTTER AND PLUNGER, METHOD- BLOT 1.6 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Calibrated magnification: 52911 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 70

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Processing

EM softwareName: SPIDER / Category: 3D reconstruction
CTF correctionDetails: PHASE-FLIPPING EACH PARTICLE
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: ICOSAHEDRAL / Resolution: 11.5 Å / Num. of particles: 18681 / Nominal pixel size: 1.4 Å / Actual pixel size: 1.323 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1863. (DEPOSITION ID: 7817).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID-BODY FIT REFINEMENT PROTOCOL--X-RAY
RefinementHighest resolution: 11.5 Å
Refinement stepCycle: LAST / Highest resolution: 11.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6290 0 0 0 6290

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