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- PDB-8oh4: Subtomogram averaging structure of cofilactin filament inside mic... -

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Basic information

Entry
Database: PDB / ID: 8oh4
TitleSubtomogram averaging structure of cofilactin filament inside microtubule lumen of Drosophila S2 cell protrusion.
Components
  • Actin-5C
  • Cofilin/actin-depolymerizing factor homolog
KeywordsCONTRACTILE PROTEIN / Cytoskeleton / Filament / Actin / Cofilactin
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 / VEGFA-VEGFR2 Pathway / Cell-extracellular matrix interactions ...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 / 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 / actomyosin contractile ring assembly / Clathrin-mediated endocytosis / actin filament fragmentation / border follicle cell migration / compound eye development / centrosome separation / sperm individualization / UCH proteinases / establishment of planar polarity / epithelial structure maintenance / brahma complex / maintenance of protein location in cell / actin filament severing / Ino80 complex / actin filament depolymerization / tube formation / RSC-type complex / regulation of lamellipodium assembly / SWI/SNF complex / lamellipodium assembly / female gonad development / mitotic cytokinesis / actin filament polymerization / axonogenesis / positive regulation of protein secretion / actin filament organization / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / nuclear matrix / actin filament binding / actin cytoskeleton / actin binding / cytoskeleton / hydrolase activity / chromatin remodeling / regulation of transcription by RNA polymerase II / nucleoplasm / ATP binding / cytoplasm / cytosol
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)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 16.5 Å
AuthorsVentura Santos, C. / Carter, A.P.
Funding support United Kingdom, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 10, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Actin-5C
B: Actin-5C
C: Actin-5C
I: Cofilin/actin-depolymerizing factor homolog
D: Actin-5C
J: Cofilin/actin-depolymerizing factor homolog
E: Actin-5C
K: Cofilin/actin-depolymerizing factor homolog
F: Actin-5C
L: Cofilin/actin-depolymerizing factor homolog
G: Actin-5C
M: Cofilin/actin-depolymerizing factor homolog
H: Actin-5C
N: Cofilin/actin-depolymerizing factor homolog


Theoretical massNumber of molelcules
Total (without water)432,37114
Polymers432,37114
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Actin-5C


Mass: 41160.980 Da / Num. of mol.: 8
Mutation: 6 N-terminal residues (MCDEEV) were removed. Residues 42-50 (QGVMVGMGC) were removed
Source method: isolated from a natural source / Source: (natural) Drosophila (fruit flies) / Cell line: S2 cells
References: UniProt: P10987, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#2: Protein
Cofilin/actin-depolymerizing factor homolog / Protein D61 / Protein twinstar


Mass: 17180.529 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Drosophila (fruit flies) / Cell line: S2 cells / References: UniProt: P45594

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Cofilactin filament inside the microtubule lumen of induced Drosophila S2 cell protrusion
Type: CELL / Entity ID: all / Source: NATURAL
Source (natural)Organism: Drosophila (fruit flies)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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.
Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 198.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 6000 nm / Nominal defocus min: 2500 nm
Image recordingElectron dose: 3 e/Å2 / Avg electron dose per subtomogram: 120 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
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.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2SerialEM4.1.0betaimage acquisition
11RELION3.1classification
12RELION3.13D reconstruction
CTF correctionDetails: CTF estimation was performed in WARP. CTF correction was performed in Relion 3.1.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 16.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3801
Details: helical symmetry (-162 twist, 29A rise) was applied during 3D classification but not during refinements.
Symmetry type: POINT
EM volume selectionNum. of tomograms: 58 / Num. of volumes extracted: 7549
Atomic model buildingDetails: Drosophila 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 ...Details: Drosophila 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.

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