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- EMDB-13781: cryo iDPC-STEM structure recorded with CSA 4.0 -

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Basic information

Entry
Database: EMDB / ID: EMD-13781
Titlecryo iDPC-STEM structure recorded with CSA 4.0
Map datacryo iDPC-STEM mao recorded with CSA 4.0
Sample
  • Virus: Tobacco mosaic virus
    • Protein or peptide: Capsid proteinCapsid
    • RNA: RNA (5'-R(P*GP*AP*A)-3')
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 / identical protein binding / Capsid protein
Function and homology information
Biological speciesTobacco mosaic virus (vulgare) / Tobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsSachse C / Leidl ML
Funding support Germany, 1 items
OrganizationGrant numberCountry
Helmholtz Association Germany
CitationJournal: Nat Methods / Year: 2022
Title: Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution.
Authors: Ivan Lazić / Maarten Wirix / Max Leo Leidl / Felix de Haas / Daniel Mann / Maximilian Beckers / Evgeniya V Pechnikova / Knut Müller-Caspary / Ricardo Egoavil / Eric G T Bosch / Carsten Sachse /
Abstract: In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently ...In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.
History
DepositionOct 26, 2021-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13781.png

Downloads & links

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Map

FileDownload / File: emd_13781.map.gz / Format: CCP4 / Size: 143.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo iDPC-STEM mao recorded with CSA 4.0
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.98 Å/pix.
x 150 pix.
= 147. Å		0.98 Å/pix.
x 500 pix.
= 490. Å		0.98 Å/pix.
x 500 pix.
= 490. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.98 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.014
Minimum - Maximum-0.044910975 - 0.061708406
Average (Standard dev.)0.00034799258 (±0.0029184807)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00176
Dimensions500500150
Spacing500500150
CellA: 490.0 Å / B: 490.0 Å / C: 147.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13781_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map1

Fileemd_13781_half_map_1.map
Annotationhalf-map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map2

Fileemd_13781_half_map_2.map
Annotationhalf-map2
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
  • Virus: Tobacco mosaic virus
    • Protein or peptide: Capsid proteinCapsid
    • RNA: RNA (5'-R(P*GP*AP*A)-3')

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Tobacco mosaic virus
Molecular weightTheoretical: 131 kDa/nm

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Macromolecule #1: Capsid protein

MacromoleculeName: Capsid protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tobacco mosaic virus (vulgare) / Strain: vulgare
Molecular weightTheoretical: 17.091998 KDa
SequenceString:
SYSITTPSQF VFLSSAWADP IELINLCTNA LGNQFQTQQA RTVVQRQFSE VWKPSPQVTV RFPDSDFKVY RYNAVLDPLV TALLGAFDT RNRIIEVENQ ANPTTAETLD ATRRVDDATV AIRSAINNLI VELIRGTGSY NRSSFESSSG LVWT

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Macromolecule #2: RNA (5'-R(P*GP*AP*A)-3')

MacromoleculeName: RNA (5'-R(P*GP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Tobacco mosaic virus (vulgare)
Molecular weightTheoretical: 958.66 Da
SequenceString:
GAA

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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

Concentration90 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: blot force of +10 and a duration for blotting of 10 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm
DetailsiDPC-STEM
Image recordingFilm or detector model: OTHER / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.408 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.03 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 2224
DetailsiDPC-STEM
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4udv

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7q2r:
cryo iDPC-STEM structure recorded with CSA 4.0

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