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- EMDB-9730: Cryo-EM structure of Flock House Virus eluted particle -

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Basic information

Entry
Database: EMDB / ID: EMD-9730
TitleCryo-EM structure of Flock House Virus eluted particle
Map dataCryo-EM structure of Flock House Virus eluted particle
Sample
  • Virus: Flock house virus
    • Protein or peptide: CAPSID PROTEIN BETA
Function / homology
Function and homology information


nodavirus endopeptidase / symbiont entry into host cell via permeabilization of host membrane / T=3 icosahedral viral capsid / aspartic-type endopeptidase activity / proteolysis / metal ion binding
Similarity search - Function
Peptidase A6, nodavirus coat protein / Peptidase A6 family / Viral coat protein subunit
Similarity search - Domain/homology
Capsid protein alpha
Similarity search - Component
Biological speciesFlock house virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsBanerjee M / Azad K
Funding support India, 1 items
OrganizationGrant numberCountry
Department of Science & Technology (India)EMR/2015/000644 India
CitationJournal: J Virol / Year: 2019
Title: Structural Dynamics of Nonenveloped Virus Disassembly Intermediates.
Authors: Kimi Azad / Manidipa Banerjee /
Abstract: The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive ...The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive understanding of how stable, uniform icosahedrons disassemble remains elusive, mainly due to the complexities involved in isolating transient intermediates. We utilized incremental heating to systematically characterize the disassembly pathway of a model nonenveloped virus and identified an intriguing link between virus maturation and disassembly. Further, we isolated and characterized two intermediates by cryo-electron microscopy and three-dimensional reconstruction, without imposing icosahedral symmetry. The first intermediate displayed a series of major, asymmetric alterations, whereas the second showed that the act of genome release, through the 2-fold axis, is actually confined to a small section on the capsid. Our study thus presents a comprehensive structural analysis of nonenveloped virus disassembly and emphasizes the asymmetric nature of programmed conformational changes. Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by nonenveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates: "eluted particles" and "puffed particles" of an insect nodavirus, Flock House virus (FHV). Cryo-electron microscopy and three-dimensional reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.
History
DepositionNov 20, 2018-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 2019-
UpdateMar 11, 2020-
Current statusMar 11, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.14
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.14
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6itb
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6itf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9730.png

Downloads & links

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Map

FileDownload / File: emd_9730.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Flock House Virus eluted particle
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.74 Å/pix.
x 256 pix.
= 445.619 Å		1.74 Å/pix.
x 256 pix.
= 445.619 Å		1.74 Å/pix.
x 256 pix.
= 445.619 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.7407 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14 / Movie #1: 0.14
Minimum - Maximum-0.19812593 - 0.38223028
Average (Standard dev.)0.013937887 (±0.036496904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 445.6192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.740699218751.740699218751.74069921875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z445.619445.619445.619
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.1980.3820.014

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Supplemental data

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Sample components

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Entire : Flock house virus

EntireName: Flock house virus
Components
  • Virus: Flock house virus
    • Protein or peptide: CAPSID PROTEIN BETA

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Supramolecule #1: Flock house virus

SupramoleculeName: Flock house virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Purified wildtype Flock House Virus heated to 70 degC
NCBI-ID: 12287 / Sci species name: Flock house virus / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Costelytra zealandica (beetle)
Host systemOrganism: Drosophila melanogaster (fruit fly)
Molecular weightExperimental: 9 MDa
Virus shellShell ID: 1 / Diameter: 300.0 Å / T number (triangulation number): 3

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Macromolecule #1: CAPSID PROTEIN BETA

MacromoleculeName: CAPSID PROTEIN BETA / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: nodavirus endopeptidase
Source (natural)Organism: Flock house virus
Molecular weightTheoretical: 39.367355 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: MVNNNRPRRQ RAQRVVVTTT QTAPVPQQNV PRNGRRRRNR TRRNRRRVRG MNMAALTRLS QPGLAFLKCA FAPPDFNTDP GKGIPDRFE GKVVSRKDVL NQSISFTAGQ DTFILIAPTP GVAYWSASVP AGTFPTSATT FNPVNYPGFT SMFGTTSTSR S DQVSSFRY ...String:
MVNNNRPRRQ RAQRVVVTTT QTAPVPQQNV PRNGRRRRNR TRRNRRRVRG MNMAALTRLS QPGLAFLKCA FAPPDFNTDP GKGIPDRFE GKVVSRKDVL NQSISFTAGQ DTFILIAPTP GVAYWSASVP AGTFPTSATT FNPVNYPGFT SMFGTTSTSR S DQVSSFRY ASMNVGIYPT SNLMQFAGSI TVWKCPVKLS TVQFPVATDP ATSSLVHTLV GLDGVLAVGP DNFSESFIKG VF SQSACNE PDFEFNDILE GIQTLPPANV SLGSTGQPFT MDSGAEATSG VVGWGNMDTI VIRVSAPEGA VNSAILKAWS CIE YRPNPN AMLYQFGHDS PPLDEVALQE YRTVARSLPV AVIAAQN

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7 / Component - Concentration: 50.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 284 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 3 seconds before plunging.
DetailsPurified wildtype Flock House Virus heated to 70 degC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 2835 / Average exposure time: 5.0 sec. / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 61905 / Details: Manual selection
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Startup modelType of model: OTHER
Details: 3D initial model was generated de novo from the 2D particles in RELION
Final reconstructionNumber classes used: 30 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 58918
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 40
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4ftb

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 181
Output model

PDB-6itb:
Icosahedral asymmetric unit (iASU) model of the well-refined part of FHV eluted particle

PDB-6itf:
Icosahedral asymmetric unit (iASU) model of the less refined, coarse part of FHV eluted particle

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