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- EMDB-13669: In situ cryo-electron tomogram of a podosome in a primary human m... -

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Basic information

Entry
Database: EMDB / ID: EMD-13669
TitleIn situ cryo-electron tomogram of a podosome in a primary human macrophage (Volta phase plate, bin 4)
Map dataIn situ cryo-electron tomogram of a podosome in a primary human macrophage (Volta phase plate, bin 4)
Sample
  • Cell: Podosome assembled at the basal membrane of a human primary monocyte-derived macrophage
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsJasnin M
Funding support Germany, 3 items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)RGP0035/2016 Germany
Foundation for Medical Research (France)FRM DEQ2016 0334894 Germany
German Research Foundation (DFG)ANR-DFG JA-3038/2-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Elasticity of podosome actin networks produces nanonewton protrusive forces.
Authors: Marion Jasnin / Jordan Hervy / Stéphanie Balor / Anaïs Bouissou / Amsha Proag / Raphaël Voituriez / Jonathan Schneider / Thomas Mangeat / Isabelle Maridonneau-Parini / Wolfgang Baumeister ...Authors: Marion Jasnin / Jordan Hervy / Stéphanie Balor / Anaïs Bouissou / Amsha Proag / Raphaël Voituriez / Jonathan Schneider / Thomas Mangeat / Isabelle Maridonneau-Parini / Wolfgang Baumeister / Serge Dmitrieff / Renaud Poincloux /
Abstract: Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin ...Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of a few tens of nanonewtons, in a range similar to that evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.
History
DepositionOct 4, 2021-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13669.png

Downloads & links

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Map

FileDownload / File: emd_13669.map.gz / Format: CCP4 / Size: 572.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ cryo-electron tomogram of a podosome in a primary human macrophage (Volta phase plate, bin 4)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
16.84 Å/pix.
x 175 pix.
= 2947. Å		16.84 Å/pix.
x 926 pix.
= 15593.84 Å		16.84 Å/pix.
x 926 pix.
= 15593.84 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: 16.84 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3.0 - 3.0
Average (Standard dev.)0.86956364 (±0.1259773)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0041
Dimensions926926175
Spacing926926175
CellA: 15593.84 Å / B: 15593.84 Å / C: 2947.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Podosome assembled at the basal membrane of a human primary monoc...

EntireName: Podosome assembled at the basal membrane of a human primary monocyte-derived macrophage
Components
  • Cell: Podosome assembled at the basal membrane of a human primary monocyte-derived macrophage

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Supramolecule #1: Podosome assembled at the basal membrane of a human primary monoc...

SupramoleculeName: Podosome assembled at the basal membrane of a human primary monocyte-derived macrophage
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
GridModel: Quantifoil / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
Cryo protectantNo
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 5000 nm / Focused ion beam - Final thickness: 300 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsBi-directional tilt series were acquired with the Volta phase plate from -30 to +68 degrees and -32 to -50 degrees with a tilt increment of 2 degrees
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 2.0 sec. / Average electron dose: 2.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.27 µm / Nominal defocus min: 0.27 µm / Nominal magnification: 33000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 60

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