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- EMDB-6639: Cryo-electron microscopy of a deletion mutant (d385-399) of Flock... -

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Basic information

Entry
Database: EMDB / ID: EMD-6639
TitleCryo-electron microscopy of a deletion mutant (d385-399) of Flock House virus VLP
Map dataReconstruction of deletion mutant (d385-399) Flock House virus VLP
Sample
  • Sample: Virus-like particle of deletion mutant (d385-399) of Flock House virus
  • Virus: Flock house virus
KeywordsFlock House virus / VLP / gamma
Biological speciesFlock house virus
Methodsingle particle reconstruction / cryo EM / Resolution: 14.5 Å
AuthorsBajaj S / Dey D / Kumar M / Banerjee M
CitationJournal: J Mol Biol / Year: 2016
Title: Non-Enveloped Virus Entry: Structural Determinants and Mechanism of Functioning of a Viral Lytic Peptide.
Authors: Saumya Bajaj / Debajit Dey / Rohan Bhukar / Mohit Kumar / Manidipa Banerjee /
Abstract: In the absence of lipid envelopes and associated fusion proteins, non-enveloped viruses employ membrane lytic peptides to breach the limiting membranes of host cells. Although several of these lytic ...In the absence of lipid envelopes and associated fusion proteins, non-enveloped viruses employ membrane lytic peptides to breach the limiting membranes of host cells. Although several of these lytic peptides have been identified and characterized, their manner of deployment and interaction with host membranes remains unclear in most cases. We are using the gamma peptide of Flock House Virus (FHV), a model non-enveloped virus, to understand the mechanistic details of non-enveloped virus interaction with host cell membranes. We utilized a combination of biophysical assays, molecular dynamics simulation studies, and single-particle cryo-electron microscopy to elucidate the functional and structural determinants for membrane penetration by gamma in context of the FHV capsid. Although the amphipathic, helical N-terminal region of gamma (γ1) was previously thought to be the membrane-penetrating module, with the C-terminal region having a supporting role in correct structural positioning of γ1, we demonstrate that the C terminus of gamma directly participates in membrane penetration. Our studies suggest that full-length gamma, including the hydrophobic C terminus, forms an alpha-helical hairpin motif, and any disruption in this motif drastically reduces its functionality, in spite of the correct positioning of amphipathic γ1 in the virus capsid. Taken together, our data suggest that the most effective module for membrane disruption is a pentameric unit of full-length gamma, released from the virus, which associates with membranes via both N- and C-terminal ends.
History
DepositionApr 29, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseDec 28, 2016-
UpdateDec 28, 2016-
Current statusDec 28, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6639.tif

Downloads & links

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Map

FileDownload / File: emd_6639.map.gz / Format: CCP4 / Size: 210.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of deletion mutant (d385-399) Flock House virus VLP
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-0.78284472 - 1.65642786
Average (Standard dev.)0.07824882 (±0.2857343)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-192-192-192
Dimensions384384384
Spacing384384384
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z384.000384.000384.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-192-192-192
NC/NR/NS384384384
D min/max/mean-0.7831.6560.078

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Supplemental data

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Sample components

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Entire : Virus-like particle of deletion mutant (d385-399) of Flock House virus

EntireName: Virus-like particle of deletion mutant (d385-399) of Flock House virus
Components
  • Sample: Virus-like particle of deletion mutant (d385-399) of Flock House virus
  • Virus: Flock house virus

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Supramolecule #1000: Virus-like particle of deletion mutant (d385-399) of Flock House virus

SupramoleculeName: Virus-like particle of deletion mutant (d385-399) of Flock House virus
type: sample / ID: 1000
Oligomeric state: 180 molecules of capsid protein make up one particle
Number unique components: 1
Molecular weightTheoretical: 9 MDa

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Supramolecule #1: Flock house virus

SupramoleculeName: Flock house virus / type: virus / ID: 1
Details: Residues 385-399 of the capsid protein have been deleted
NCBI-ID: 12287 / Sci species name: Flock house virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Costelytra zealandica (beetle) / synonym: INVERTEBRATES
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf21 / Recombinant plasmid: pBacPAK9
Molecular weightTheoretical: 9 MDa
Virus shellShell ID: 1 / Name: Alpha / Diameter: 336 Å / T number (triangulation number): 3

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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

Concentration2.5 mg/mL
BufferpH: 7.4 / Details: 50 mM HEPES, 5mM CaCl2
GridDetails: 300 mesh copper, Quantifoil R2/2 grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK IV
Method: Blot for 3 seconds at a blot force of 0 before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 77 K / Max: 97 K / Average: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 80,000x magnification
DateJan 8, 2016
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 1199 / Average electron dose: 10 e/Å2 / Bits/pixel: 2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 82
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 20199
DetailsParticles were boxed using the "Swarm mode" of e2boxer.py. Initial model generated by imposing icosahedral symmetry, and 3D refinement using e2refine_easy.py
FSC plot (resolution estimation)

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