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- EMDB-53197: Dual-axis CSTET tomogram of mitochondria from MFF-/- U2-OS cells ... -

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Basic information

Entry
Database: EMDB / ID: EMD-53197
TitleDual-axis CSTET tomogram of mitochondria from MFF-/- U2-OS cells under fission-inducing conditions
Map dataDual-axis cryo-STET tomogram of MFF-/- U-2 OS cells under fission-inducing conditions
Sample
  • Cell: Human bone osteosarcoma cells, U2-OS
KeywordsMitochondrial morphology / whole cell tomography / cryo-STEM tomography / CSTET / UNKNOWN FUNCTION
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsKirchweger P / Wolf SG / Elbaum M
Funding support Austria, Israel, European Union, 4 items
OrganizationGrant numberCountry
Austrian Science FundJ4449-B Austria
Israel Science Foundation1696/18 Israel
European Union (EU)IMpaCT (grant no. 857203)European Union
European Research Council (ERC)101055413European Union
Citation
Journal: J Cell Sci / Year: 2025
Title: Snapshots of mitochondrial fission imaged by cryo-scanning transmission electron tomography.
Authors: Peter Kirchweger / Sharon Grayer Wolf / Neta Varsano / Tali Dadosh / Guenter P Resch / Michael Elbaum /
Abstract: Mitochondria undergo constant remodeling via fission, fusion, extension and degradation. Fission, in particular, depends on the accumulation of mitochondrial fission factor (MFF) and subsequent ...Mitochondria undergo constant remodeling via fission, fusion, extension and degradation. Fission, in particular, depends on the accumulation of mitochondrial fission factor (MFF) and subsequent recruitment of the dynamin-related protein DRP1 (also known as DNM1L). We used cryo-scanning transmission electron tomography (cryo-STET) to investigate mitochondrial morphologies in MFF mutant (MFF-/-) mouse embryonic fibroblast (MEF) cells in ATP-depleting conditions that normally induce fission. The capability of cryo-STET to image through the cytoplasmic volume to a depth of 1 µm facilitated visualization of intact mitochondria and their surroundings. We imaged changes in mitochondrial morphology and cristae structure, as well as contacts with the endoplasmic reticulum (ER), degradative organelles and the cytoskeleton at stalled fission sites. We found disruption of the outer mitochondrial membrane at contact sites with the ER and degradative organelles at sites of mitophagy. We identified fission sites where the inner mitochondrial membrane is already separated while the outer membrane is still continuous. Although MFF is a general fission factor, these observations demonstrate that mitochondrial fission can proceed to the final stage in its absence. The use of cryo-STET allays concerns about the loss of structures due to sample thinning required for tomography using cryo-transmission electron microscopy.
#1: Journal: Biorxiv / Year: 2024
Title: Snapshots of Mitochondrial Fission Imaged by Cryo-Scanning Transmission Electron Tomography
Authors: Kirchweger P / Wolf SG / Varsano N / Dadosh T / Resch GP / Elbaum M
History
DepositionMar 17, 2025-
Header (metadata) releaseApr 16, 2025-
Map releaseApr 16, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53197.png

Downloads & links

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Map

FileDownload / File: emd_53197.map.gz / Format: CCP4 / Size: 2.4 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationDual-axis cryo-STET tomogram of MFF-/- U-2 OS cells under fission-inducing conditions
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
20.42 Å/pix.
x 307 pix.
= 6268.94 Å		20.42 Å/pix.
x 2047 pix.
= 41799.738 Å		20.42 Å/pix.
x 2047 pix.
= 41799.738 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 20.42 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-12996.0 - 32767.0
Average (Standard dev.)4771.015999999999622 (±398.532069999999976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin11-156
Dimensions20472047307
Spacing20472047307
CellA: 41799.74 Å / B: 41799.74 Å / C: 6268.94 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Human bone osteosarcoma cells, U2-OS

EntireName: Human bone osteosarcoma cells, U2-OS
Components
  • Cell: Human bone osteosarcoma cells, U2-OS

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Supramolecule #1: Human bone osteosarcoma cells, U2-OS

SupramoleculeName: Human bone osteosarcoma cells, U2-OS / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Details: DMEM growth medium
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 310.15 K / Instrument: LEICA EM GP
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Home Made / Diameter: 15 nm

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 122 / Average exposure time: 50.0 sec. / Average electron dose: 1.05 e/Å2
Details: Images were recorded on a Fischione HAADF detector in BrightField mode (described in Kirchweger et al., 2023).
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Nominal magnification: 29000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION
Software:
Namedetails
IMOD (ver. 4.12.10)
PRIISM/IVE (ver. 4.7.2)deconvolved using core2_decon

Details: We used a custom Python script for deconvolution / Number images used: 122
CTF correctionDetails: CSTET needs no CTF correction / Type: NONE

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