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- EMDB-16877: Subtomogram averaging structure of cofilactin filament inside mic... -

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Basic information

Entry
Database: EMDB / ID: EMD-16877
TitleSubtomogram averaging structure of cofilactin filament inside microtubule lumen of Drosophila S2 cell protrusion.
Map dataMasked map at 16.5 A resolution. Sharpened with a B-factor of -500 A2.
Sample
  • Cell: Cofilactin filament inside the microtubule lumen of induced Drosophila S2 cell protrusion
    • Protein or peptide: Actin-5C
    • Protein or peptide: Cofilin/actin-depolymerizing factor homolog
Function / homology
Function and homology information


establishment of imaginal disc-derived wing hair orientation / establishment of ommatidial planar polarity / imaginal disc-derived leg segmentation / meiotic cytokinesis / Gap junction degradation / Formation of annular gap junctions / EPHB-mediated forward signaling / EPH-ephrin mediated repulsion of cells / Recycling pathway of L1 / VEGFA-VEGFR2 Pathway ...establishment of imaginal disc-derived wing hair orientation / establishment of ommatidial planar polarity / imaginal disc-derived leg segmentation / meiotic cytokinesis / Gap junction degradation / Formation of annular gap junctions / EPHB-mediated forward signaling / EPH-ephrin mediated repulsion of cells / Recycling pathway of L1 / VEGFA-VEGFR2 Pathway / : / Cell-extracellular matrix interactions / RHOBTB2 GTPase cycle / RHOF GTPase cycle / MAP2K and MAPK activation / Platelet degranulation / RHO GTPases Activate WASPs and WAVEs / Regulation of actin dynamics for phagocytic cup formation / compound eye morphogenesis / DNA Damage Recognition in GG-NER / mushroom body development / rhabdomere development / actin filament fragmentation / UCH proteinases / actomyosin contractile ring assembly / Clathrin-mediated endocytosis / border follicle cell migration / compound eye development / centrosome separation / sperm individualization / establishment of planar polarity / epithelial structure maintenance / brahma complex / maintenance of protein location in cell / tube formation / actin filament severing / Ino80 complex / actin filament depolymerization / regulation of lamellipodium assembly / lamellipodium assembly / female gonad development / mitotic cytokinesis / actin filament polymerization / axonogenesis / actin filament organization / positive regulation of protein secretion / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / cell morphogenesis / nuclear matrix / actin filament binding / actin cytoskeleton / actin binding / cytoskeleton / hydrolase activity / chromatin remodeling / nucleoplasm / ATP binding / cytosol / cytoplasm
Similarity search - Function
ADF/Cofilin / Actin-depolymerising factor homology domain / Cofilin/tropomyosin-type actin-binding protein / ADF-H domain profile. / Actin depolymerisation factor/cofilin -like domains / ADF-H/Gelsolin-like domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site ...ADF/Cofilin / Actin-depolymerising factor homology domain / Cofilin/tropomyosin-type actin-binding protein / ADF-H domain profile. / Actin depolymerisation factor/cofilin -like domains / ADF-H/Gelsolin-like domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Actin-5C / Cofilin/actin-depolymerizing factor homolog
Similarity search - Component
Biological speciesDrosophila (fruit flies) / fruit flies (fruit flies)
Methodsubtomogram averaging / cryo EM / Resolution: 16.5 Å
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 20, 2023-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateMay 10, 2023-
Current statusMay 10, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16877.png

Downloads & links

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Map

FileDownload / File: emd_16877.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked map at 16.5 A resolution. Sharpened with a B-factor of -500 A2.
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.83 Å/pix.
x 256 pix.
= 724.48 Å		2.83 Å/pix.
x 256 pix.
= 724.48 Å		2.83 Å/pix.
x 256 pix.
= 724.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00337
Minimum - Maximum-0.008817553 - 0.011251742
Average (Standard dev.)-3.3504755e-06 (±0.00048764705)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 724.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16877_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: Unmasked map at 16.5 A resolution. Sharpened with...

Fileemd_16877_additional_1.map
AnnotationUnmasked map at 16.5 A resolution. Sharpened with a B-factor of -500 A2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2.

Fileemd_16877_half_map_1.map
AnnotationHalf map 2.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1.

Fileemd_16877_half_map_2.map
AnnotationHalf map 1.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cofilactin filament inside the microtubule lumen of induced Droso...

EntireName: Cofilactin filament inside the microtubule lumen of induced Drosophila S2 cell protrusion
Components
  • Cell: Cofilactin filament inside the microtubule lumen of induced Drosophila S2 cell protrusion
    • Protein or peptide: Actin-5C
    • Protein or peptide: Cofilin/actin-depolymerizing factor homolog

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Supramolecule #1: Cofilactin filament inside the microtubule lumen of induced Droso...

SupramoleculeName: Cofilactin filament inside the microtubule lumen of induced Drosophila S2 cell protrusion
type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Drosophila (fruit flies)

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Macromolecule #1: Actin-5C

MacromoleculeName: Actin-5C / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: fruit flies (fruit flies)
Molecular weightTheoretical: 41.16098 KDa
SequenceString: AALVVDNGSG MCKAGFAGDD APRAVFPSIV GRPRHQGVMV GMGQKDSYVG DEAQSKRGIL TLKYPIEHGI VTNWDDMEKI WHHTFYNEL RVAPEEHPVL LTEAPLNPKA NREKMTQIMF ETFNTPAMYV AIQAVLSLYA SGRTTGIVLD SGDGVSHTVP I YEGYALPH ...String:
AALVVDNGSG MCKAGFAGDD APRAVFPSIV GRPRHQGVMV GMGQKDSYVG DEAQSKRGIL TLKYPIEHGI VTNWDDMEKI WHHTFYNEL RVAPEEHPVL LTEAPLNPKA NREKMTQIMF ETFNTPAMYV AIQAVLSLYA SGRTTGIVLD SGDGVSHTVP I YEGYALPH AILRLDLAGR DLTDYLMKIL TERGYSFTTT AEREIVRDIK EKLCYVALDF EQEMATAASS SSLEKSYELP DG QVITIGN ERFRCPEALF QPSFLGMEAC GIHETTYNSI MKCDVDIRKD LYANTVLSGG TTMYPGIADR MQKEITALAP STM KIKIIA PPERKYSVWI GGSILASLST FQQMWISKQE YDESGPSIVH RKCF

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Macromolecule #2: Cofilin/actin-depolymerizing factor homolog

MacromoleculeName: Cofilin/actin-depolymerizing factor homolog / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: fruit flies (fruit flies)
Molecular weightTheoretical: 17.180529 KDa
SequenceString:
MASGVTVSDV CKTTYEEIKK DKKHRYVIFY IRDEKQIDVE TVADRNAEYD QFLEDIQKCG PGECRYGLFD FEYMHQCQGT SESSKKQKL FLMSWCPDTA KVKKKMLYSS SFDALKKSLV GVQKYIQATD LSEASREAVE EKLRATDRQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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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: Quantifoil R3.5/1 Au200 grids were glow discharged for 30s at 20 - 30 mA and subsequently coated with 0.25 ug/mL Concanavalin Aater for 1 - 16 h at 37 degrees Celcius.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 198.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 6.0 µm / Nominal defocus min: 2.5 µm
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 electron dose: 3.0 e/Å2
Details: Data was collected on Gatan K2 summit (2.952 A/pixel) and Gatan K3 summit (2.659 A/pixel) with 3 degree increments (3 e/A2 dose per tilt). The total dose was between 118 and 122 e/A2.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 58 / Number images used: 7549
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: helical symmetry (-162 twist, 29A rise) was applied during 3D classification but not during refinements.
Number subtomograms used: 3801
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsDrosophila cofilin and actin structures were predicted as a complex with Alphafold 2-Multimer. Actin from the predicted cofilin-actin complex was iteratively aligned with 8 actin subunits in the chicken cofilactin PDB model 5yU8. Two cofilin moieties at the pointed end were removed. Side chains were truncated. The model was not refined.
Output model

PDB-8oh4:
Subtomogram averaging structure of cofilactin filament inside microtubule lumen of Drosophila S2 cell protrusion.

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