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- EMDB-18493: Cryo-electron tomogram of lift-out lamella from cell-derived extr... -

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Basic information

Entry
Database: EMDB / ID: EMD-18493
TitleCryo-electron tomogram of lift-out lamella from cell-derived extracellular matrix (example 4)
Map dataTomogram acquired on cryo-lift-out lamella of a cell-derived matrix showing mainly extracellular structures such as collagen.
Sample
  • Cell: Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derived matrix from human telomerase-immortalized foreskin fibroblasts (TIFFs)
KeywordsExtracellular matrix / cell-derived matrix / collagen / PROTEIN FIBRIL
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsZens B / Faessler F / Hansen J / Hauschild R / Datler J / Hodirnau V-V / Zheden V / Alanko J / Sixt MK / Schur FKM
Funding support Austria, United States, European Union, 4 items
OrganizationGrant numberCountry
Austrian Science FundP33367 Austria
Austrian Science FundE435 Austria
Chan Zuckerberg InitiativeDAF2021-234754 United States
European Research Council (ERC)724373European Union
CitationJournal: J Cell Biol / Year: 2024
Title: Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix.
Authors: Bettina Zens / Florian Fäßler / Jesse M Hansen / Robert Hauschild / Julia Datler / Victor-Valentin Hodirnau / Vanessa Zheden / Jonna Alanko / Michael Sixt / Florian K M Schur /
Abstract: The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in ...The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.
History
DepositionSep 20, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateApr 3, 2024-
Current statusApr 3, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18493.png

Downloads & links

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Map

FileDownload / File: emd_18493.map.gz / Format: CCP4 / Size: 313.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram acquired on cryo-lift-out lamella of a cell-derived matrix showing mainly extracellular structures such as collagen.
Voxel sizeX=Y=Z: 17.096 Å
Density
Minimum - Maximum-7.576888 - 5.1969466
Average (Standard dev.)-0.46805674 (±0.2982876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions720510224
Spacing510720224
CellA: 8718.96 Å / B: 12309.12 Å / C: 3829.5042 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derive...

EntireName: Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derived matrix from human telomerase-immortalized foreskin fibroblasts (TIFFs)
Components
  • Cell: Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derived matrix from human telomerase-immortalized foreskin fibroblasts (TIFFs)

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Supramolecule #1: Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derive...

SupramoleculeName: Cryo-lamella prepared by lift-out cryo-FIB milling of cell-derived matrix from human telomerase-immortalized foreskin fibroblasts (TIFFs)
type: cell / ID: 1 / Parent: 0
Details: Cell-derived matrix was generated by growing TIFF cells on EM grids over 14 days of growth time.
Source (natural)Organism: Homo sapiens (human) / Organ: Skin / Tissue: Foreskin

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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 / Component - Concentration: 10.0 Percent / Component - Name: Dextran / Details: 10% Dextran in 0.1M Phosphate Buffer, pH 7.4
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 / Details: ELMO Glow Discharge unit
VitrificationCryogen name: NITROGEN
DetailsCell-derived matrix obtained from TIFF-cells.
High pressure freezingInstrument: OTHER
Details: High pressure freezing carriers were 3.0mm in diameter and 0.5mm thick. One carrier was flat, the second carrier had a central cavity of 2.0mm diameter and 0.02mm depth. Carriers were coated ...Details: High pressure freezing carriers were 3.0mm in diameter and 0.5mm thick. One carrier was flat, the second carrier had a central cavity of 2.0mm diameter and 0.02mm depth. Carriers were coated in 1-hexadecene prior to high pressure freezing.. The value given for _em_high_pressure_freezing.instrument is Bal-tec HPM010. This is not in a list of allowed values {'BAL-TEC HPM 010', 'EMS-002 RAPID IMMERSION FREEZER', 'LEICA EM PACT', 'LEICA EM PACT2', 'LEICA EM HPM100', 'OTHER'} so OTHER is written into the XML file.
Cryo protectant10% Dextran (high MW)
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 3600 / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 250
Focused ion beam - Details: A block of sample was extracted from the sample bulk by cryo-lift out FIB milling, with a thickness of 5000-8000nm. It was attached to a second grid, where it was then ...Focused ion beam - Details: A block of sample was extracted from the sample bulk by cryo-lift out FIB milling, with a thickness of 5000-8000nm. It was attached to a second grid, where it was then milled down to 200-250nm thickness step by step. Lamellae were milled in consecutive steps with 1nA (down to 3000nm thickness), 0.5nA (2000nm thickness), 0.1nA (1000nm), 50pA (500nm) and 30pA (200nm). See associated publication for details.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos II. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 8.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 83.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 0.5 sec. / Average electron dose: 2.137 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION
Details: Reconstructed with AreTomo (version 1.3.4., Feb22) and processed with IsoNet (version 0.2).
Number images used: 61

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