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- EMDB-26214: F-actin from neuronal growth cone filopodium -

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Basic information

Entry
Database: EMDB / ID: EMD-26214
TitleF-actin from neuronal growth cone filopodium
Map dataSubtomogram average of filamentous actin from a neuronal growth cone filopodium.
Sample
  • Organelle or cellular component: Filamentous actin from a neuronal growth cone filopodium
KeywordsCytoskeleton / Polymer / Filament / Actin / STRUCTURAL PROTEIN
Biological speciesRattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM / Resolution: 20.2 Å
AuthorsHylton RK / Swulius MT
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Cofilactin filaments regulate filopodial structure and dynamics in neuronal growth cones.
Authors: Ryan K Hylton / Jessica E Heebner / Michael A Grillo / Matthew T Swulius /
Abstract: Cofilin is best known for its ability to sever actin filaments and facilitate cytoskeletal recycling inside of cells, but at higher concentrations in vitro, cofilin stabilizes a more flexible, hyper- ...Cofilin is best known for its ability to sever actin filaments and facilitate cytoskeletal recycling inside of cells, but at higher concentrations in vitro, cofilin stabilizes a more flexible, hyper-twisted state of actin known as "cofilactin". While this filament state is well studied, a structural role for cofilactin in dynamic cellular processes has not been observed. With a combination of cryo-electron tomography and fluorescence imaging in neuronal growth cones, we observe that filopodial actin filaments switch between a fascin-linked and a cofilin-decorated state, and that cofilactin is associated with a variety of dynamic events within filopodia. The switch to cofilactin filaments occurs in a graded fashion and correlates with a decline in fascin cross-linking within the filopodia, which is associated with curvature in the bundle. Our tomographic data reveal that the hyper-twisting of actin from cofilin binding leads to a rearrangement of filament packing, which largely excludes fascin from the base of filopodia. Our results provide mechanistic insight into the fundamentals of cytoskeletal remodeling inside of confined cellular spaces, and how the interplay between fascin and cofilin regulates the dynamics of searching filopodia.
History
DepositionFeb 16, 2022-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26214.png

Downloads & links

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Map

FileDownload / File: emd_26214.map.gz / Format: CCP4 / Size: 1.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of filamentous actin from a neuronal growth cone filopodium.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.31 Å/pix.
x 192 pix.
= 826.752 Å		4.31 Å/pix.
x 48 pix.
= 206.688 Å		4.31 Å/pix.
x 48 pix.
= 206.688 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 4.306 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.723
Minimum - Maximum-3.939249 - 6.7720814
Average (Standard dev.)-0.016271792 (±1.0294733)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin16170
Dimensions4848192
Spacing4848192
CellA: 206.68802 Å / B: 206.68802 Å / C: 826.7521 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Filamentous actin from a neuronal growth cone filopodium

EntireName: Filamentous actin from a neuronal growth cone filopodium
Components
  • Organelle or cellular component: Filamentous actin from a neuronal growth cone filopodium

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Supramolecule #1: Filamentous actin from a neuronal growth cone filopodium

SupramoleculeName: Filamentous actin from a neuronal growth cone filopodium
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Cryo-electron tomography was performed on filopodia of neurons growing on EM grids. The map is a result from subtomogram averaging of ~83 nm-long filament sections (1603 particles total) ...Details: Cryo-electron tomography was performed on filopodia of neurons growing on EM grids. The map is a result from subtomogram averaging of ~83 nm-long filament sections (1603 particles total) from an f-actin bundle in a single filopodium.
Source (natural)Organism: Rattus norvegicus (Norway rat) / Strain: Spague Dawley / Organ: Brain / Tissue: Hippocampus / Location in cell: Growth cone filopodia

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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
Details: No buffer was used. The cells were grown on EM grids in Neurobasal media with B-27 supplement (2%) and Penicillin/Streptomycin (1%) or NbActiv4 (with 1% Penicillin/Streptomycin) neuronal cell culture media.
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV
Details: 3 uL of 10 nm gold fiducials were added on top of the cells prior to blotting or freezing. Grids were then blotted by hand from behind (the non-cell surface) for 2 seconds before plunge-freezing..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 0.9 sec. / Average electron dose: 2.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 26000
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 reconstructionApplied symmetry - Helical parameters - Δz: 27.6 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 20.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 1603
ExtractionNumber tomograms: 1 / Number images used: 1603 / Reference model: Non-refined average of all particles. / Method: Filaments picked by hand. / Software - Name: Dynamo
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial modelPDB ID:

6t1y


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT

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