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- EMDB-19425: Low-dose cryo-electron ptychographic reconstruction of apoferriti... -

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Basic information

Entry
Database: EMDB / ID: EMD-19425
TitleLow-dose cryo-electron ptychographic reconstruction of apoferritin recorded with CSA of 4.0 mrad
Map datasharpened map
Sample
  • Complex: Apoferritin
KeywordsApoFerritin / METAL BINDING PROTEIN
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsMohammed I / Stalhberg H / Kucukoglu B
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation200021_200628 Switzerland
CitationJournal: 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.
History
DepositionJan 17, 2024-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19425.png

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Map

FileDownload / File: emd_19425.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 154 pix.
= 231. Å		1.5 Å/pix.
x 154 pix.
= 231. Å		1.5 Å/pix.
x 154 pix.
= 231. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
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Contour LevelBy AUTHOR: 0.544
Minimum - Maximum-2.0024998 - 3.2870882
Average (Standard dev.)-0.0036878418 (±0.24388203)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions154154154
Spacing154154154
CellA=B=C: 231.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19425_msk_1.map
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Half map: #2

Fileemd_19425_half_map_1.map
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Half map: #1

Fileemd_19425_half_map_2.map
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Sample components

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Entire : Apoferritin

EntireName: Apoferritin
Components
  • Complex: Apoferritin

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Supramolecule #1: Apoferritin

SupramoleculeName: Apoferritin / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 480 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / 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: OTHER / Imaging mode: OTHER / Nominal defocus max: 1.7 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailspy4DSTEM cryoSPARC
Startup modelType of model: NONE / Details: ab-initio
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 11552
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1) / Details: point group symmetry applied:O
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1) / Details: point group symmetry applied:O
FSC plot (resolution estimation)

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