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- EMDB-18475: Cryo-electron tomogram of an induced S2 cell protrusion. The cell... -

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Basic information

Entry
Database: EMDB / ID: EMD-18475
TitleCryo-electron tomogram of an induced S2 cell protrusion. The cell was treated with 2uM thapsigargin (5h) and with 2.5uM Cytochalasin D (2h).
Map dataCryo-electron tomogram of an induced S2 cell protrusion after treatment with 2.5uM CytD (2h) and 2um thapsigargin (5h).
Sample
  • Cell: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
Keywordsmicrotubules / protrusion / STRUCTURAL PROTEIN
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)MRC_UP_A025_1011 United Kingdom
Wellcome Trust210711/Z/18/Z United Kingdom
CitationJournal: EMBO Rep / 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 identities of these objects and mechanisms for their accumulation have not been ...Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila 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 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, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference 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
DepositionSep 18, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18475.png

Downloads & links

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Map

FileDownload / File: emd_18475.map.gz / Format: CCP4 / Size: 2.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomogram of an induced S2 cell protrusion after treatment with 2.5uM CytD (2h) and 2um thapsigargin (5h).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10.74 Å/pix.
x 376 pix.
= 4038.24 Å		10.74 Å/pix.
x 1440 pix.
= 15465.6 Å		10.74 Å/pix.
x 1022 pix.
= 10976.279 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: 10.74 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.60988736 - 0.43933672
Average (Standard dev.)0.000000000000655 (±0.0399006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401022376
Spacing10221440376
CellA: 10976.279 Å / B: 15465.6 Å / C: 4038.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cytochalasin D-induced protrusion of a Drosophila S2 cell.

EntireName: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
Components
  • Cell: Cytochalasin D-induced protrusion of a Drosophila S2 cell.

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Supramolecule #1: Cytochalasin D-induced protrusion of a Drosophila S2 cell.

SupramoleculeName: Cytochalasin D-induced protrusion of a Drosophila S2 cell.
type: cell / ID: 1 / Parent: 0
Details: Cells were treated with 2uM thapsigargin for 5h and with 2.5uM Cytochalasin D for 2h prior to vitrification.
Source (natural)Organism: Drosophila melanogaster (fruit fly) / Strain: Schneider 2 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
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: Grids were glow discharged for 30s at 20mA and then coated with 0.25ug/mL Concanavalin A. Grids were washed twice with PBS before plating cells.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI Solutions / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 0.56 sec. / Average electron dose: 2.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe tomogram has been binned by 4 and deconvolved in Warp.
Final reconstructionSoftware - Name: Warp (ver. 1.0.9) / Number images used: 41

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