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- EMDB-19885: Low-dose cryo-electron ptychographic reconstruction of TMV record... -

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Basic information

Entry
Database: EMDB / ID: EMD-19885
TitleLow-dose cryo-electron ptychographic reconstruction of TMV recorded with CSA of 6.1 mrad
Map data
Sample
  • Virus: Tobacco mosaic virus
KeywordsCapsid / Tobacco Mosaic Virus / VIRUS
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 6.4 Å
AuthorsMohammed I / Stalhberg H / Kucukoglu B
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation200021_200628 Switzerland
Citation
Journal: Nat Commun / Year: 2024
Title: Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution.
Authors: Berk Küçükoğlu / Inayathulla Mohammed / Ricardo C Guerrero-Ferreira / Stephanie M Ribet / Georgios Varnavides / Max Leo Leidl / Kelvin Lau / Sergey Nazarov / Alexander Myasnikov / ...Authors: Berk Küçükoğlu / Inayathulla Mohammed / Ricardo C Guerrero-Ferreira / Stephanie M Ribet / Georgios Varnavides / Max Leo Leidl / Kelvin Lau / Sergey Nazarov / Alexander Myasnikov / Massimo Kube / Julika Radecke / Carsten Sachse / Knut Müller-Caspary / Colin Ophus / Henning Stahlberg /
Abstract: Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron ...Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.
#1: Journal: To Be Published
Title: Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution
Authors: Mohammed I / Stalhberg H / Kucukoglu B
History
DepositionMar 19, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19885.png

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Map

FileDownload / File: emd_19885.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 340 pix.
= 396.406 Å		1.17 Å/pix.
x 340 pix.
= 396.406 Å		1.17 Å/pix.
x 340 pix.
= 396.406 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1659 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0207
Minimum - Maximum-0.06152712 - 0.07650287
Average (Standard dev.)0.000039933526 (±0.0040540462)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 396.406 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19885_msk_1.map
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Half map: #1

Fileemd_19885_half_map_1.map
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Half map: #2

Fileemd_19885_half_map_2.map
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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
Molecular weightTheoretical: 40.0 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: DECTRIS ELA (1k x 0.5k) / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: DIFFRACTION / Cs: 0.1 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 1.38 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.036 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.1) / Number images used: 2120
Segment selectionNumber selected: 4686
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 4.1)
FSC plot (resolution estimation)

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