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- EMDB-1525: Structure of Thermusphage P23-77 using electron cryomicroscopy an... -

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Basic information

Entry
Database: EMDB / ID: EMD-1525
TitleStructure of Thermusphage P23-77 using electron cryomicroscopy and three-dimensional image reconstruction
Map dataThis is a 14 A resolution cryo-EM reconstruction of Thermusphage P23-77
Sample
  • Sample: Thermusphage P23-77
  • Virus: Thermus phage P23-77 (virus)
KeywordsIcosahedral thermophilic bacterial virus
Biological speciesThermus phage P23-77 (virus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 14.0 Å
AuthorsJaatinen ST / Happonen LJ / Laurinmaki P / Butcher SJ / Bamford DH
CitationJournal: Virology / Year: 2008
Title: Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus.
Authors: S T Jaatinen / L J Happonen / P Laurinmäki / S J Butcher / D H Bamford /
Abstract: Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were ...Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families.
History
DepositionJun 11, 2008-
Header (metadata) releaseJun 12, 2008-
Map releaseJan 26, 2010-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7000
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 7000
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1525.map.gz / Format: CCP4 / Size: 120.1 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationThis is a 14 A resolution cryo-EM reconstruction of Thermusphage P23-77
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour Level1: 6480.0 / Movie #1: 7000
Minimum - Maximum-16509.0 - 32443.0
Average (Standard dev.)-34.956800000000001 (±3684.760000000000218)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions401401401
Spacing401401401
CellA=B=C: 1122.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z2.82.82.8
M x/y/z401401401
origin x/y/z0.0000.0000.000
length x/y/z1122.8001122.8001122.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS401401401
D min/max/mean-16509.00032443.000-34.957

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Supplemental data

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Sample components

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Entire : Thermusphage P23-77

EntireName: Thermusphage P23-77
Components
  • Sample: Thermusphage P23-77
  • Virus: Thermus phage P23-77 (virus)

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Supramolecule #1000: Thermusphage P23-77

SupramoleculeName: Thermusphage P23-77 / type: sample / ID: 1000
Details: The sample was purified on a sucrose gradient, and stored at 28 C prior to plunging.
Oligomeric state: 1 / Number unique components: 1

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Supramolecule #1: Thermus phage P23-77

SupramoleculeName: Thermus phage P23-77 / type: virus / ID: 1 / Name.synonym: Thermusphage P23-77 / NCBI-ID: 668994 / Sci species name: Thermus phage P23-77 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No / Syn species name: Thermusphage P23-77
Host (natural)Organism: Thermus thermophilus (bacteria) / synonym: BACTERIA(EUBACTERIA)
Virus shellShell ID: 1 / Diameter: 780 Å / T number (triangulation number): 28

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20 mM Tris-HCl pH 7.5, 1mM MgCl2, 0.1 mM CaCl2
StainingType: NEGATIVE
Details: Vitrified. Grids were blotted for roughly one second before being plunged into liquid ethane.
GridDetails: 400 mesh copper grid, Quantifoil R2/2 holey
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Guillotine
Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding 3 microliters of the sample is held in place at the bottom of a plunger by the means of fine ...Method: A small vial of ethane is placed inside a larger liquid nitrogen reservoir. The grid holding 3 microliters of the sample is held in place at the bottom of a plunger by the means of fine tweezers. When the liquid ethane is ready, a piece of filter paper is then pressed against the sample to blot off excess buffer, sufficient to leave a thin layer on the grid. The filter paper is removed, and the plunger is allowed to drop into the liquid ethane. Once the grid enters the liquid ethane, the sample is rapidly frozen, and the grid is transferred under liquid nitrogen to a storage box immersed in liquid nitrogen for later use in the microscope.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49300 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
DetailsLow dose conditions.
DateMar 19, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 25
Details: Images were scanned at 7 microns, and binned by 2 to 14 microns step size.

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PFT, POR, EM3DR2, P3DR / Number images used: 880
DetailsThe particles were selected using an automatic selection program (ETHAN).

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