- 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
Title
3D reconstruction of TMV at 4.5A from film micrographs using Frealix
Map data
TMV 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 species
Tobacco mosaic virus
Method
helical reconstruction / cryo EM / Resolution: 4.5 Å
Journal: 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
Deposition
Apr 22, 2015
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Header (metadata) release
Apr 29, 2015
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Map release
Apr 29, 2015
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Update
Apr 29, 2015
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Current status
Apr 29, 2015
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_6325.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
TMV 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.
Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Method: Blot for 2 seconds before plunging
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Electron microscopy
Microscope
FEI TECNAI F30
Date
Nov 22, 2005
Image recording
Category: 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
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Filaments were processed using Frealix assuming constant helical parameters and without continuity restraints on the helical parameters.
Final reconstruction
Applied 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.
CTF correction
Details: Defocus estimated for each helical subunit
FSC plot (resolution estimation)
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