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- EMDB-6325: 3D reconstruction of TMV at 4.5A from film micrographs using Frealix -

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Basic information

Entry
Database: EMDB / ID: EMD-6325
Title3D reconstruction of TMV at 4.5A from film micrographs using Frealix
Map dataTMV map filtered using a negative B-factor, with amplitude matching against density derived from fitted atomic coordinates, a figure-of-merit filter, and subsequent helical symmetrization, as detailed in the primary citation.
Sample
  • Sample: Tobacco mosaic virus
  • Virus: Tobacco mosaic virus
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsRohou A / Grigorieff N
CitationJournal: J Mol Biol / Year: 2007
Title: High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus.
Authors: Carsten Sachse / James Z Chen / Pierre-Damien Coureux / M Elizabeth Stroupe / Marcus Fändrich / Nikolaus Grigorieff /
Abstract: The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be ...The treatment of helical objects as a string of single particles has become an established technique to resolve their three-dimensional (3D) structure using electron cryo-microscopy. It can be applied to a wide range of helical particles such as viruses, microtubules and helical filaments. We have made improvements to this approach using Tobacco Mosaic Virus (TMV) as a test specimen and obtained a map from 210,000 asymmetric units at a resolution better than 5 A. This was made possible by performing a full correction of the contrast transfer function of the microscope. Alignment of helical segments was helped by constraints derived from the helical symmetry of the virus. Furthermore, symmetrization was implemented by multiple inclusions of symmetry-related views in the 3D reconstruction. We used the density map to build an atomic model of TMV. The model was refined using a real-space refinement strategy that accommodates multiple conformers. The atomic model shows significant deviations from the deposited model for the helical form of TMV at the lower-radius region (residues 88 to 109). This region appears more ordered with well-defined secondary structure, compared with the earlier helical structure. The RNA phosphate backbone is sandwiched between two arginine side-chains, stabilizing the interaction between RNA and coat protein. A cluster of two or three carboxylates is buried in a hydrophobic environment isolating it from neighboring subunits. These carboxylates may represent the so-called Caspar carboxylates that form a metastable switch for viral disassembly. Overall, the observed differences suggest that the new model represents a different, more stable state of the virus, compared with the earlier published model.
History
DepositionApr 22, 2015-
Header (metadata) releaseApr 29, 2015-
Map releaseApr 29, 2015-
UpdateApr 29, 2015-
Current statusApr 29, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6325.gif

Downloads & links

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Map

FileDownload / File: emd_6325.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTMV map filtered using a negative B-factor, with amplitude matching against density derived from fitted atomic coordinates, a figure-of-merit filter, and subsequent helical symmetrization, as detailed in the primary citation.
Voxel sizeX=Y=Z: 1.163 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-2.5420177 - 3.56391788
Average (Standard dev.)-0.00057129 (±0.67606211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 232.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1631.1631.163
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z232.600232.600232.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-2.5423.564-0.001

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Supplemental data

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Supplemental map: 3drec.map

File3drec.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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

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Supramolecule #1000: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: sample / ID: 1000 / Oligomeric state: Helical assembly / Number unique components: 1

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Sci species strain: vulgare / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Nicotiana tabacum (common tobacco) / synonym: PLANTAE(HIGHER PLANTS)
Virus shellShell ID: 1 / Name: Virus shell 1 / Diameter: 180 Å

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration2.5 mg/mL
BufferDetails: Phosphate, 5 mM EDTA
GridDetails: Quantifoil grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.86 µm / Nominal defocus min: 1.688 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateNov 22, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7.0 µm / Number real images: 7 / Average electron dose: 15 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Defocus estimated for each helical subunit
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.39259 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.0318 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: OTHER / Software - Name: Frealix
Details: Only data up to 5.5 Angstrom were used during refinement in the final rounds. In earlier rounds, this threshold was set to a lower resolution.
DetailsFilaments were processed using Frealix assuming constant helical parameters and without continuity restraints on the helical parameters.
FSC plot (resolution estimation)

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