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- EMDB-16720: Tomogram of an induced protrusion of a Drosophila S2 cell with fi... -

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Basic information

Entry
Database: EMDB / ID: EMD-16720
TitleTomogram of an induced protrusion of a Drosophila S2 cell with filaments inside the microtubule lumen.
Map dataDeconvolved tomogram (binned by four) of an induced protrusion from Drosophila S2 cells with multiple filaments inside the microtubule lumen. From Dataset 7.
Sample
  • Cell: Tomogram of an induced protrusion of a Drosophila S2 cell with filaments inside the microtubule lumen
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
DepositionFeb 17, 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_16720.png

Downloads & links

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Map

FileDownload / File: emd_16720.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 Drosophila S2 cells with multiple filaments inside the microtubule lumen. From Dataset 7.
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-0.40263182 - 0.35335198
Average (Standard dev.)-3.9809464e-13 (±0.04133351)
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 Drosophila S2 cell with fi...

EntireName: Tomogram of an induced protrusion of a Drosophila S2 cell with filaments inside the microtubule lumen
Components
  • Cell: Tomogram of an induced protrusion of a Drosophila S2 cell with filaments inside the microtubule lumen

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Supramolecule #1: Tomogram of an induced protrusion of a Drosophila S2 cell with fi...

SupramoleculeName: Tomogram of an induced protrusion of a Drosophila S2 cell with filaments inside the microtubule lumen
type: cell / ID: 1 / Parent: 0
Details: Cells were treated with 2.5 uM Cytochalasin D for 4 hours and with 2 uM thapsigargin for 5 hours before vitrification. Example tomogram from dataset 7.
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
GridModel: Quantifoil / Material: GOLD / Mesh: 200
Details: Grids were glow discharged for 30s at 20mA and then coated with 0.25 ug/mL Concanavalin A for at least 2 hours at 37 degrees.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298.15 K
DetailsCells were treated with 2.5 uM Cytochalasin D for 4 hours and with 2 uM thapsigargin for 5 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 BIOQUANTUM (6k x 4k) / Average electron dose: 3.0 e/Å2
Details: Data was collected at a pixel size of 2.659 A/pixel with a total dose of 121.54 A/e^2.
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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