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- EMDB-19489: Tobacco mosaic virus from scanning transmission electron microsco... -

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Basic information

Entry
Database: EMDB / ID: EMD-19489
TitleTobacco mosaic virus from scanning transmission electron microscopy at CSA=2.0 mrad
Map datasharp map
Sample
  • Virus: Tobacco mosaic virus
Keywordstobacco mosaic virus / VIRUS
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 5.4 Å
AuthorsMann D / Filopoulou A / Sachse C
Funding support Germany, 1 items
OrganizationGrant numberCountry
Helmholtz AssociationPoF 5352 Germany
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2024
Title: VitroJet: new features and case studies.
Authors: Rene J M Henderikx / Daniel Mann / Aušra Domanska / Jing Dong / Saba Shahzad / Behnam Lak / Aikaterini Filopoulou / Damian Ludig / Martin Grininger / Jeffrey Momoh / Elina Laanto / Hanna M ...Authors: Rene J M Henderikx / Daniel Mann / Aušra Domanska / Jing Dong / Saba Shahzad / Behnam Lak / Aikaterini Filopoulou / Damian Ludig / Martin Grininger / Jeffrey Momoh / Elina Laanto / Hanna M Oksanen / Kyrylo Bisikalo / Pamela A Williams / Sarah J Butcher / Peter J Peters / Bart W A M M Beulen /
Abstract: Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before ...Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.
History
DepositionJan 25, 2024-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19489.png

Downloads & links

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Map

FileDownload / File: emd_19489.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharp map
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 256 pix.
= 320. Å		1.25 Å/pix.
x 256 pix.
= 320. Å		1.25 Å/pix.
x 256 pix.
= 320. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.6762507 - 1.2316965
Average (Standard dev.)0.0068476996 (±0.14015952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19489_msk_1.map
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Half map: half map b

Fileemd_19489_half_map_1.map
Annotationhalf map b
Projections & Slices
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Half map: half map a

Fileemd_19489_half_map_2.map
Annotationhalf map a
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Sample components

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Entire : Tobacco mosaic virus

EntireName: Tobacco mosaic virus
Components
  • Virus: Tobacco mosaic virus

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Virus type: VIRION / Virus isolate: OTHER / Virus enveloped: Yes / Virus empty: No

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration33 mg/mL
BufferpH: 7 / Details: distilled water (MilliQ)
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: OTHER
Details: VitroJet pins were cleaned with detergent and 70% EtOH in an ultrasonicator followed by 1 min drying under nitrogen stream (for 5 minutes each). TMV was pin printed at 4 degC chamber ...Details: VitroJet pins were cleaned with detergent and 70% EtOH in an ultrasonicator followed by 1 min drying under nitrogen stream (for 5 minutes each). TMV was pin printed at 4 degC chamber temperature with a 70 micrometer spiral, 15 micrometer standoff with a velocity of 5 mm per second. The value given for _em_vitrification.instrument is CRYOSOL VITROJET. This is not in a list of allowed values {'HOMEMADE PLUNGER', 'FEI VITROBOT MARK III', 'FEI VITROBOT MARK II', 'FEI VITROBOT MARK I', 'GATAN CRYOPLUNGE 3', 'LEICA PLUNGER', 'LEICA EM GP', 'REICHERT-JUNG PLUNGER', 'LEICA EM CPC', 'OTHER', 'FEI VITROBOT MARK IV', 'SPOTITON', 'EMS-002 RAPID IMMERSION FREEZER', 'LEICA KF80'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Detailsin focus iDPC-STEM measurement using Velox software
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 103 / Average electron dose: 35.0 e/Å2 / Details: segmented TFS Panther iDPC-STEM detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.0 µm / Calibrated defocus min: 0.0 µm / Illumination mode: SPOT SCAN / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 240000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsiDPC images were generated in TFS Velox software and highpass filtered with a gaussian highpass filter with a width of 251 angstroms
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.03 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 5.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 13644
Startup modelType of model: OTHER / Details: featureless cylinder (diameter 180 angstroms)
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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