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- PDB-6h2b: Structure of the Macrobrachium rosenbergii Nodavirus -

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Basic information

Entry
Database: PDB / ID: 6h2b
TitleStructure of the Macrobrachium rosenbergii Nodavirus
ComponentsCapsid proteinCapsid
KeywordsVIRUS LIKE PARTICLE / Nodavirus / Capsid / Virion
Function / homologyViral coat protein subunit / Capsid protein alpha
Function and homology information
Biological speciesMacrobrachium rosenbergii nodavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsHo, K.H. / Gabrielsen, M. / Beh, P.L. / Kueh, C.L. / Thong, Q.X. / Streetley, J. / Tan, W.S. / Bhella, D.
Funding support United Kingdom, Malaysia, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UU_12014/7 United Kingdom
UPM GP-IPS/2016/9509400 Malaysia
CitationJournal: PLoS Biol / Year: 2018
Title: Structure of the Macrobrachium rosenbergii nodavirus: A new genus within the Nodaviridae?
Authors: Kok Lian Ho / Mads Gabrielsen / Poay Ling Beh / Chare Li Kueh / Qiu Xian Thong / James Streetley / Wen Siang Tan / David Bhella /
Abstract: Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many ...Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV.
History
DepositionJul 13, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 31, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,9319
Polymers124,6913
Non-polymers2406
Water0
1
A: Capsid protein
B: Capsid protein
C: Capsid protein
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)7,495,875540
Polymers7,481,447180
Non-polymers14,428360
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: Capsid protein
B: Capsid protein
C: Capsid protein
hetero molecules
x 5


  • icosahedral pentamer
  • 625 kDa, 15 polymers
Theoretical massNumber of molelcules
Total (without water)624,65645
Polymers623,45415
Non-polymers1,20230
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: Capsid protein
B: Capsid protein
C: Capsid protein
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 750 kDa, 18 polymers
Theoretical massNumber of molelcules
Total (without water)749,58854
Polymers748,14518
Non-polymers1,44336
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Capsid protein / Capsid


Mass: 41563.594 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macrobrachium rosenbergii nodavirus / Gene: CP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: X2FE19
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca

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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: Macrobrachium rosenbergii nodavirus / Type: VIRUS / Details: Capsid protein was expressed in Sf9 cells / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Macrobrachium rosenbergii nodavirus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Macrobrachium rosenbergii
Virus shellName: Capsid / Diameter: 400 nm / Triangulation number (T number): 3
Buffer solutionpH: 7.4 / Details: 20 mM HEPES, 100 mM NaCl pH 7.4
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: VLPs were deposited onto a continuous carbon film that had been floated onto a quantifoil holey carbon film (R2/2)

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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 / Calibrated magnification: 47170 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 36 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2459
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20 / Used frames/image: 2-20

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELION2.1CTF correction
7Cootmodel fitting
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELIONclassification
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 60939 / Details: Automated particle picking in Relion 2.1
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40883 / Algorithm: BACK PROJECTION / Details: Standard Relion workflow for icosahedral particle. / Symmetry type: POINT
Atomic model buildingB value: 140 / Protocol: AB INITIO MODEL / Space: REAL
Details: Model built largely ab initio, following docking of a homology model based on PDB 4LLF. The homology model matched in two strands at residues - 104-135 and 232-243. This served as the ...Details: Model built largely ab initio, following docking of a homology model based on PDB 4LLF. The homology model matched in two strands at residues - 104-135 and 232-243. This served as the starting point for manual model building. The model was then subjected to real space refinement using Phenix.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00778004
ELECTRON MICROSCOPYf_angle_d0.843140750
ELECTRON MICROSCOPYf_dihedral_angle_d8.81631614
ELECTRON MICROSCOPYf_chiral_restr0.0596381
ELECTRON MICROSCOPYf_plane_restr0.00412283

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