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- EMDB-5844: Subtomogram average of a virus-associated pyramid -

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Basic information

Entry
Database: EMDB / ID: EMD-5844
TitleSubtomogram average of a virus-associated pyramid
Map dataReconstruction of a virus associated pyramid
Sample
  • Sample: Reconstruction of a virus-associated pyramid induced by overexpressing the viral protein PVAP in E. coli
  • Protein or peptide: protein forming virus-associated pyramids
KeywordsVAP / archaeovirus / archaea / SIRV2 / Sulfolobus islandicus / viral egress
Function / homology: / membrane => GO:0016020 / Uncharacterized protein
Function and homology information
Biological speciesSulfolobus islandicus rod-shaped virus 2
Methodsubtomogram averaging / cryo EM
AuthorsDaum B / Kuehlbrandt W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.
Authors: Bertram Daum / Tessa E F Quax / Martin Sachse / Deryck J Mills / Julia Reimann / Özkan Yildiz / Sabine Häder / Cosmin Saveanu / Patrick Forterre / Sonja-Verena Albers / Werner Kühlbrandt ...Authors: Bertram Daum / Tessa E F Quax / Martin Sachse / Deryck J Mills / Julia Reimann / Özkan Yildiz / Sabine Häder / Cosmin Saveanu / Patrick Forterre / Sonja-Verena Albers / Werner Kühlbrandt / David Prangishvili /
Abstract: Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. ...Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.
History
DepositionDec 19, 2013-
Header (metadata) releaseFeb 5, 2014-
Map releaseFeb 19, 2014-
UpdateApr 23, 2014-
Current statusApr 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 172.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 172.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5844_1.jpg

emd_5844_2.tif

Downloads & links

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Map

FileDownload / File: emd_5844.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a virus associated pyramid
Voxel sizeX=Y=Z: 8.663 Å
Density
Contour LevelBy AUTHOR: 172.400000000000006 / Movie #1: 172.4
Minimum - Maximum161.271331790000005 - 179.423156739999996
Average (Standard dev.)169.777954099999988 (±1.47277522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 866.3 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.6638.6638.663
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z866.300866.300866.300
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean161.271179.423169.778

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Supplemental data

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Sample components

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Entire : Reconstruction of a virus-associated pyramid induced by overexpre...

EntireName: Reconstruction of a virus-associated pyramid induced by overexpressing the viral protein PVAP in E. coli
Components
  • Sample: Reconstruction of a virus-associated pyramid induced by overexpressing the viral protein PVAP in E. coli
  • Protein or peptide: protein forming virus-associated pyramids

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Supramolecule #1000: Reconstruction of a virus-associated pyramid induced by overexpre...

SupramoleculeName: Reconstruction of a virus-associated pyramid induced by overexpressing the viral protein PVAP in E. coli
type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: protein forming virus-associated pyramids

MacromoleculeName: protein forming virus-associated pyramids / type: protein_or_peptide / ID: 1 / Name.synonym: P98, PVAP
Details: The gene encoding PVAP was heterologously overexpressed in E. coli to incorporate pyramid-shaped multimers of PVAP into the plasma membrane.
Oligomeric state: Multimer / Recombinant expression: Yes
Source (natural)Organism: Sulfolobus islandicus rod-shaped virus 2 / Location in cell: Plasma membrane
Molecular weightExperimental: 10 KDa / Theoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta(DE3)pLys / Recombinant plasmid: pSA4
SequenceUniProtKB: Uncharacterized protein / GO: GO: 0951408

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7 / Details: 50 mM Tris, 300 mM NaCl
GridDetails: Quantifoil 300 mesh R2/2, glow-discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 10 % / Instrument: HOMEMADE PLUNGER
Method: Blot for 3-5 seconds from the side where the sample was applied.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 16870 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 10.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 27500
Specialist opticsEnergy filter - Name: Gatan Tridium GIF
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60.73 ° / Tilt series - Axis1 - Max angle: 59.20 °
TemperatureMin: 89 K / Max: 96 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 40,000 times magnification
DateMar 12, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Bits/pixel: 32
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: PEET, IMOD / Number subtomograms used: 57

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