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- EMDB-15666: Actin filament from lamellipodia of mouse embryonic fibroblasts -

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Basic information

Entry
Database: EMDB / ID: EMD-15666
TitleActin filament from lamellipodia of mouse embryonic fibroblasts
Map data
Sample
  • Cell: Mouse embryonic fibroblast
KeywordsActin filament / STRUCTURAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 14.7 Å
AuthorsWen-Lu C / Ohad M
Funding support Switzerland, European Union, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
European Research Council (ERC)European Union
CitationJournal: Commun Biol / Year: 2022
Title: A network of mixed actin polarity in the leading edge of spreading cells.
Authors: Wen-Lu Chung / Matthias Eibauer / Wenhong Li / Rajaa Boujemaa-Paterski / Benjamin Geiger / Ohad Medalia /
Abstract: Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes. The actin cytoskeleton is a central driver and regulator of cellular ...Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes. The actin cytoskeleton is a central driver and regulator of cellular dynamics, that produces membrane-protrusions such as lamellipodia and filopodia. Here, we examined actin organization in expanding lamellipodia during early stages of cell spreading. To gain insight into the 3D actin organization, we plated fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant (~10%) population of actin filaments, are oriented with their barbed-ends towards the cell center. Our results provide structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.
History
DepositionAug 24, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15666.png

Downloads & links

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Map

FileDownload / File: emd_15666.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
2.21 Å/pix.
x 160 pix.
= 353.04 Å		2.21 Å/pix.
x 160 pix.
= 353.04 Å		2.21 Å/pix.
x 160 pix.
= 353.04 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00614
Minimum - Maximum-0.018896619 - 0.035344753
Average (Standard dev.)0.00001044843 (±0.0027574347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 353.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15666_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #2

Fileemd_15666_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Mouse embryonic fibroblast

EntireName: Mouse embryonic fibroblast
Components
  • Cell: Mouse embryonic fibroblast

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Supramolecule #1: Mouse embryonic fibroblast

SupramoleculeName: Mouse embryonic fibroblast / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 2.46 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.5 µm / Nominal defocus min: 3.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.6 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.7 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 14.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number subtomograms used: 149617
ExtractionNumber tomograms: 58 / Number images used: 212537
Final angle assignmentType: ANGULAR RECONSTITUTION

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