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- PDB-6i5a: Tobacco Mosaic Virus -

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Basic information

Entry
Database: PDB / ID: 6i5a
TitleTobacco Mosaic Virus
ComponentsCapsid proteinCapsid
KeywordsVIRUS / TMV / helical virus / coat protein / plant pathogen
Function / homology
Function and homology information


helical viral capsid / structural molecule activity / identical protein binding
Similarity search - Function
Tobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein superfamily / Virus coat protein (TMV like) / Four Helix Bundle (Hemerythrin (Met), subunit A) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Biological speciesTobacco mosaic virus
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsSong, B. / Flegler, V. / Makbul, C. / Rasmussen, T. / Bottcher, B.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation359471283 Germany
CitationJournal: Ultramicroscopy / Year: 2019
Title: Capabilities of the Falcon III detector for single-particle structure determination.
Authors: Boyuan Song / Julian Lenhart / Vanessa Judith Flegler / Cihan Makbul / Tim Rasmussen / Bettina Böttcher /
Abstract: Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the ...Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e/Px² and integrating mode capability at exposure rates above 10 e/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode.
History
DepositionNov 13, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1May 1, 2019Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Capsid protein


Theoretical massNumber of molelcules
Total (without water)17,6371
Polymers17,6371
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area8950 Å2
MethodPISA

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Components

#1: Protein Capsid protein / Capsid / Coat protein


Mass: 17636.621 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tobacco mosaic virus (strain vulgare) / Strain: vulgare / References: UniProt: P69687

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Tobacco mosaic virus / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightValue: 40 MDa / Experimental value: NO
Source (natural)Organism: Tobacco mosaic virus
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Nicotiana sp.
Virus shellDiameter: 180 nm
Buffer solutionpH: 7
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Quantifoil 400 mesh copper grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at medium power ...Details: Quantifoil 400 mesh copper grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at medium power with a Harrick Plasma cleaner (PDC-002). Subsequently, 3-4 ul of the sample was pipetted onto the glow discharged grids and plunge frozen in liquid Ethane with a Vitrobot IV (FEI). The sample chamber of the Vitrobot was maintained at 4C and 100% humidity. Wait time between sample application and blotting was 45 s, the drain time 0 s, the blot time 3 s and the blot force 0.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 580 nm / Calibrated defocus max: 2120 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 79 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 55 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1001
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2EPUimage acquisition
4RELIONCTF correction
7Warp8model fittingautomatic model buildung with the ARP/WARP server
9Warp8model refinementautomatic model buildung with the ARP/WARP server
10RELIONinitial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
Image processingDetails: Movies were motion corrected and exposure weighted either with motioncor2 (see tables 1-3 for details). The defocus was determined for the motion corrected and exposure weighted averages with ctffind4
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 22.037 ° / Axial rise/subunit: 1.39615 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 393692
Details: For TMV, some helices were picked manually with the helix picker implemented in relion 2.1. An initial 3D model of these segments was determined using the relion helix option with a cylinder ...Details: For TMV, some helices were picked manually with the helix picker implemented in relion 2.1. An initial 3D model of these segments was determined using the relion helix option with a cylinder as the starting reference. The aligned helical segments after 3D refinement were subjected to 2D classification without alignment. Three of the 2D class averages were selected as templates for automatic picking of the TMV segments in relion.
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91927 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 105 / Protocol: AB INITIO MODEL / Space: RECIPROCAL

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