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- EMDB-1730: 4.6 Angstrom Cryo-EM reconstruction of Tobacco Mosaic Virus from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1730
Title4.6 Angstrom Cryo-EM reconstruction of Tobacco Mosaic Virus from images recorded at 300 KeV on a 4kx4k CCD camera
Map dataB-factor scaled fully CTF corrected density map of TMV
Sample
  • Sample: Tobacco Mosaic Virus
  • Virus: Tobacco mosaic virus
  • RNA: ssRNA
KeywordsCryo-EM / single particle processing / TMV / CCD data / 300 KeV electrons
Function / homologyTobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein superfamily / Virus coat protein (TMV like) / helical viral capsid / structural molecule activity / Capsid protein
Function and homology information
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / negative staining / Resolution: 4.6 Å
AuthorsClare DK / Orlova EV
CitationJournal: J Struct Biol / Year: 2010
Title: 4.6A Cryo-EM reconstruction of tobacco mosaic virus from images recorded at 300 keV on a 4k x 4k CCD camera.
Authors: Daniel K Clare / Elena V Orlova /
Abstract: Tobacco mosaic virus (TMV) is a plant virus with a highly ordered organisation and has been described in three different structural states: As stacked disks without RNA (X-ray crystallography), as a ...Tobacco mosaic virus (TMV) is a plant virus with a highly ordered organisation and has been described in three different structural states: As stacked disks without RNA (X-ray crystallography), as a helical form with RNA (X-ray fibre diffraction) and as a second distinct helical form with RNA (cryo-EM). Here we present a structural analysis of TMV as a test object to assess the quality of cryo-EM images recorded at 300 keV on a CCD camera. The 4.6A TMV structure obtained is consistent with the previous cryo-EM structure and confirms that there is a second helical form of TMV. The structure here also shows that with a similar number of TMV segments an equivalent resolution can be achieved with a 4k CCD camera at 300 keV.
History
DepositionMay 11, 2010-
Header (metadata) releaseJun 11, 2010-
Map releaseJun 21, 2010-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2xea
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2xea
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1730.png

Downloads & links

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Map

FileDownload / File: emd_1730.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor scaled fully CTF corrected density map of TMV
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 6.1 / Movie #1: 10
Minimum - Maximum-26.271799999999999 - 46.868499999999997
Average (Standard dev.)0.551206 (±5.7358)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 248 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z248.000248.000248.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-100-100-100
NX/NY/NZ200200200
MAP C/R/S213
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-26.27246.8690.551

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Supplemental data

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Sample components

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Entire : Tobacco Mosaic Virus

EntireName: Tobacco Mosaic Virus
Components
  • Sample: Tobacco Mosaic Virus
  • Virus: Tobacco mosaic virus
  • RNA: ssRNA

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Supramolecule #1000: Tobacco Mosaic Virus

SupramoleculeName: Tobacco Mosaic Virus / type: sample / ID: 1000 / Details: Monodisperse
Oligomeric state: Homo-oligomer of TMV coat protein and ssRNA
Number unique components: 2
Molecular weightExperimental: 175 KDa / Theoretical: 175 KDa

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / Name.synonym: TMV / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Database: NCBI / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No / Syn species name: TMV
Host (natural)Organism: Nicotiana (plant) / synonym: PLANTAE(HIGHER PLANTS)
Molecular weightExperimental: 175 KDa / Theoretical: 175 KDa
Virus shellShell ID: 1 / Name: CP / Diameter: 180 Å

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Macromolecule #1: ssRNA

MacromoleculeName: ssRNA / type: rna / ID: 1 / Name.synonym: RNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Tobacco mosaic virus / synonym: TMV

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2
StainingType: NEGATIVE
Details: 3.5ul of TMV was added to R2/2 C-flat grid, blotted, then plunge frozen in liquid ethane
GridDetails: 400 mesh R2/2 c-flat grids
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Home made plunger
Method: Grids were blotted for around 2 seconds and then rapidly plunged into liquid ethane

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 78 K
Alignment procedureLegacy - Astigmatism: Corrected at 115,000 times
DateFeb 1, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 104 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 121000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 90000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particle were selected using the helical option in boxer
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.408 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.04 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, IMAGIC / Details: BPRP was used for reconstruction
CTF correctionDetails: Each particle was fully CTF corrected
Final angle assignmentDetails: 80-100 in theta and 0-22.04 in phi in 0.5 steps

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Atomic model buiding 1

Initial modelPDB ID:

2om3

SoftwareName: Chimera and Coot
DetailsProtocol: Rigid body, real space. The PDB was initially fitted using chimera, the coordinates were then refined using Coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-2xea:
4.6 ANGSTROM CRYO-EM RECONSTRUCTION OF TOBACCO MOSAIC VIRUS FROM IMAGES RECORDED AT 300 KEV ON A 4KX4K CCD CAMERA

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