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- EMDB-16811: Tomogram of an induced protrusion of a cofilin knock-down Drosoph... -

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Basic information

Entry
Database: EMDB / ID: EMD-16811
TitleTomogram of an induced protrusion of a cofilin knock-down Drosophila S2 cell with a filament inside the microtubule lumen.
Map dataDeconvolved tomogram (binned by four) of an induced protrusion from a cofilin knock-down Drosophila S2 cell. From Dataset 11 part 2.
Sample
  • Cell: Tomogram of an induced protrusion of a cofilin knock-down Drosophila S2 cell.
Biological speciesDrosophila melanogaster (fruit fly)
Methodelectron tomography / cryo EM
AuthorsVentura Santos C / Carter AP
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1011 United Kingdom
Wellcome Trust210711/Z/18/Z United Kingdom
CitationJournal: bioRxiv / Year: 2023
Title: CryoET shows cofilactin filaments inside the microtubule lumen.
Authors: Camilla Ventura Santos / Stephen L Rogers / Andrew P Carter /
Abstract: Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been ...Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
History
DepositionMar 9, 2023-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateApr 19, 2023-
Current statusApr 19, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16811.png

Downloads & links

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Map

FileDownload / File: emd_16811.map.gz / Format: CCP4 / Size: 2.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeconvolved tomogram (binned by four) of an induced protrusion from a cofilin knock-down Drosophila S2 cell. From Dataset 11 part 2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.64 Å/pix.
x 374 pix.
= 3979.36 Å		10.64 Å/pix.
x 1440 pix.
= 15321.601 Å		10.64 Å/pix.
x 1022 pix.
= 10874.08 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: 10.64 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.4719461 - 0.43866646
Average (Standard dev.)-1.7506196e-13 (±0.037161116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401022374
Spacing10221440374
CellA: 10874.08 Å / B: 15321.601 Å / C: 3979.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Tomogram of an induced protrusion of a cofilin knock-down Drosoph...

EntireName: Tomogram of an induced protrusion of a cofilin knock-down Drosophila S2 cell.
Components
  • Cell: Tomogram of an induced protrusion of a cofilin knock-down Drosophila S2 cell.

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Supramolecule #1: Tomogram of an induced protrusion of a cofilin knock-down Drosoph...

SupramoleculeName: Tomogram of an induced protrusion of a cofilin knock-down Drosophila S2 cell.
type: cell / ID: 1 / Parent: 0
Details: Cofilin levels were reduced by siRNA depletion for 7 - 8 days. Cells were then treated with 2 uM thapsigargin for 5 hours and with 2.5 uM Cytochalasin D for 2 hours before vitrification.
Source (natural)Organism: Drosophila melanogaster (fruit fly) / Strain: S2 cells

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK III
DetailsCofilin levels were reduced by siRNA depletion for 7 - 8 days. Cells were then treated with 2 uM thapsigargin for 5 hours and with 2.5 uM Cytochalasin D for 2 hours before vitrification.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI Solutions / Diameter: 10 nm

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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 K3 (6k x 4k) / Detector mode: COUNTING / Average electron dose: 3.0 e/Å2
Details: Data was collected on Gatan K3 (2.659 A/pixel) with a total dose of 111.61 e/A2.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 41

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