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- EMDB-3215: Sub-tomogram averaging of electron cryo-microscopic data taken fr... -

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Entry
Database: EMDB / ID: EMD-3215
TitleSub-tomogram averaging of electron cryo-microscopic data taken from focused-ion beam milled lamellae of nuclei of Pseudorabies virus (PrV) nuclear egress complex-expressing cells
Map dataSub-tomogram average of nuclear egress complex from vesicles observed inside nuclei of cells co-expressing PrV UL31 and UL34 by focus-ion beam milling and electron cryo-tomography.
Sample
  • Sample: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the Collection of Cell Lines in Veterinary Medicine at the FLI, Greifswald-Insel Riems, Germany)
  • Organelle or cellular component: Nuclear envelope
Keywordsalphaherpesvirinae / herpesvirus simplex / HSV-1 / pseudorabies virus / PrV / nuclear egress complex / nuclear envelope / nucleoplasmic reticulum / inner nuclear membrane / UL31 / UL34 / vesicle transport / nucleo-cytoplasmic transport / nanovesicles / cryoEM / cryoET / electron cryo-microscopy / electron cryo-tomography / cryoFIB / focused ion beam milling / FIB-SEM
Biological speciesSus scrofa (pig)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 35.0 Å
AuthorsHagen C / Siebert CA / Dent KC / Vasishtan D / Zeev Ben Mordehai T / Grange M / Klupp BG / Mettenleiter T / Gruenewald K
Citation
Journal: Cell / Year: 2015
Title: Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.
Authors: Christoph Hagen / Kyle C Dent / Tzviya Zeev-Ben-Mordehai / Michael Grange / Jens B Bosse / Cathy Whittle / Barbara G Klupp / C Alistair Siebert / Daven Vasishtan / Felix J B Bäuerlein / ...Authors: Christoph Hagen / Kyle C Dent / Tzviya Zeev-Ben-Mordehai / Michael Grange / Jens B Bosse / Cathy Whittle / Barbara G Klupp / C Alistair Siebert / Daven Vasishtan / Felix J B Bäuerlein / Juliana Cheleski / Stephan Werner / Peter Guttmann / Stefan Rehbein / Katja Henzler / Justin Demmerle / Barbara Adler / Ulrich Koszinowski / Lothar Schermelleh / Gerd Schneider / Lynn W Enquist / Jürgen M Plitzko / Thomas C Mettenleiter / Kay Grünewald /
Abstract: Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the ...Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.
#1: Journal: Cell Reports / Year: 2015
Title: Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodelling
Authors: Zeev-Ben-Mordehai T / Weberruss M / Lorenz M / Cheleski J / Hellberg T / Whittle C / El Omari K / Vasishtan D / Dent KC Harlos K / Franzke K / Hagen C / Klupp B / Antonin W / Mettenleiter TC / Gruenewald K
History
DepositionOct 26, 2015-
Header (metadata) releaseDec 23, 2015-
Map releaseDec 23, 2015-
UpdateDec 23, 2015-
Current statusDec 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.25
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.25
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3215.png

Downloads & links

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Map

FileDownload / File: emd_3215.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram average of nuclear egress complex from vesicles observed inside nuclei of cells co-expressing PrV UL31 and UL34 by focus-ion beam milling and electron cryo-tomography.
Voxel sizeX=Y=Z: 11.4 Å
Density
Contour LevelBy AUTHOR: 1.25 / Movie #1: 1.25
Minimum - Maximum-4.83183336 - 6.67973518
Average (Standard dev.)0.11689624 (±1.27687883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-630
Dimensions646464
Spacing646464
CellA=B=C: 729.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z11.411.411.4
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z729.600729.600729.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-6300
NC/NR/NS646464
D min/max/mean-4.8326.6800.117

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Supplemental data

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Sample components

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Entire : porcine epithelial-like embryonic EFN-R kidney cells stably co-ex...

EntireName: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the ...Name: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the Collection of Cell Lines in Veterinary Medicine at the FLI, Greifswald-Insel Riems, Germany)
Components
  • Sample: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the Collection of Cell Lines in Veterinary Medicine at the FLI, Greifswald-Insel Riems, Germany)
  • Organelle or cellular component: Nuclear envelope

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Supramolecule #1000: porcine epithelial-like embryonic EFN-R kidney cells stably co-ex...

SupramoleculeName: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the ...Name: porcine epithelial-like embryonic EFN-R kidney cells stably co-expressing PrV UL31 and UL34, the latter fused with GFP (cell line designated as BK/EF/UL31/34gfp catalogue No. RIE 1083 of the Collection of Cell Lines in Veterinary Medicine at the FLI, Greifswald-Insel Riems, Germany)
type: sample / ID: 1000 / Details: cellular
Oligomeric state: coat of ~500 hexamers of pUL31/pUL34 heterodimers per vesicle
Number unique components: 2

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Supramolecule #1: Nuclear envelope

SupramoleculeName: Nuclear envelope / type: organelle_or_cellular_component / ID: 1 / Name.synonym: Inner and outer nuclear membranes
Details: two proteins (pUL31 and pUL34) of pseudorabies virus (PrV) are co-expressed in the cells forming there the herpesviral nuclear egress complex lining as a coat perinuclear vesicles
Oligomeric state: heterodimer / Recombinant expression: No
Ref INTERPROdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kIPR021152 ampajax1cl asspoptrgi IPR021152i spandiv
Ref INTERPROdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kIPR007626 ampajax1cl asspoptrgi IPR007626i spandiv
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Kidney / Cell: Epithelial-like embryonic / Organelle: Nucleus / Location in cell: Nuclear mebranes
Molecular weightExperimental: 60 KDa / Theoretical: 60 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

StainingType: NEGATIVE / Details: no staining
GridDetails: glow discharged standard 3.05 mm electron microscopy 200 mesh gold grids covered with a perforated carbon foil (R2/1; Quantifoil Micro Tools GmbH, Jena, Germany); focused-ion beam milled lamella
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Instrument: HOMEMADE PLUNGER
Timed resolved state: two days of incubation (37 degree C, 5 % CO2) in plastic microscope slide growth chambers (mue-slide 2x9 well; Ibidi GmbH) before cryo-immobilization
Method: blotted manually with a bent strip of Whatman No. 1 filter paper from the non-coated grid side for 2 to 3 s immediately before vitrification by the gravity-driven plunging apparatus in a ...Method: blotted manually with a bent strip of Whatman No. 1 filter paper from the non-coated grid side for 2 to 3 s immediately before vitrification by the gravity-driven plunging apparatus in a ethane/propane mixture cooled by liquid nitrogen

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 52650 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: -6.0 µm / Nominal defocus min: -6.0 µm / Nominal magnification: 22500
Specialist opticsEnergy filter - Name: Gatan GIF 2 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -50 ° / Tilt series - Axis1 - Max angle: 52 °
Details2048 x 2048
DateApr 26, 2013
Image recordingCategory: CCD / Film or detector model: GATAN MULTISCAN / Number real images: 35 / Average electron dose: 114 e/Å2
Details: electron cryo-tomographic tilt series with 3 degree spacing
Bits/pixel: 32
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: OTHER / Software - Name: IMOD / Number subtomograms used: 300
Details'Particle' positions and orientations were initially approximated by modelling intraluminal vesicles as a set of points distributed over a sphere. After orienting particles to align their primary axes (defined as normal to the vesicle membrane) to the Y-axis, iterative 3D orientation search and translational alignment was carried out using PEET according to standard methods. Particles from each vesicle were aligned and average independently. For further information please refer to the primary citation.

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