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- PDB-9kbw: CryoEM structure of microtubule bound with GAS2-GAR domain. -

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Basic information

Entry
Database: PDB / ID: 9kbw
TitleCryoEM structure of microtubule bound with GAS2-GAR domain.
Components
  • Detyrosinated tubulin alpha-1B chain
  • Growth arrest-specific protein 2
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / Cytoskeleton
Function / homology
Function and homology information


initiation of primordial ovarian follicle growth / Caspase-mediated cleavage of cytoskeletal proteins / ovulation / regulation of Notch signaling pathway / basement membrane organization / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport ...initiation of primordial ovarian follicle growth / Caspase-mediated cleavage of cytoskeletal proteins / ovulation / regulation of Notch signaling pathway / basement membrane organization / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Resolution of Sister Chromatid Cohesion / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / EML4 and NUDC in mitotic spindle formation / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / COPI-mediated anterograde transport / antral ovarian follicle growth / stress fiber / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / regulation of cell shape / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / regulation of cell cycle / GTPase activity / apoptotic process / GTP binding / metal ion binding / membrane / cytoplasm
Similarity search - Function
GAR domain / GAR domain superfamily / Growth-Arrest-Specific Protein 2 Domain / GAR domain profile. / Growth-Arrest-Specific Protein 2 Domain / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. ...GAR domain / GAR domain superfamily / Growth-Arrest-Specific Protein 2 Domain / GAR domain profile. / Growth-Arrest-Specific Protein 2 Domain / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta chain / Growth arrest-specific protein 2 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAn, J. / Imasaki, T. / Narita, A. / Niwa, S. / Sasaki, R. / Makino, T. / Nitta, R. / Kikkawa, M.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H04762, 21H05248 Japan
CitationJournal: EMBO J / Year: 2025
Title: Dimerization of GAS2 mediates crosslinking of microtubules and F-actin.
Authors: Jiancheng An / Tsuyoshi Imasaki / Akihiro Narita / Shinsuke Niwa / Ryohei Sasaki / Tsukasa Makino / Ryo Nitta / Masahide Kikkawa /
Abstract: The spectraplakin family protein GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual ...The spectraplakin family protein GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural basis of cytoskeletal dynamics regulation by GAS2 remains unclear. In this study, we present cryo-electron microscopy structures of the GAS2 type 3 calponin homology domain (CH3) in complex with F-actin at 2.8 Å resolution, thus solving the first type CH3 domain structure bound to F-actin and confirming its actin-binding activity. We also provide the first near-atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identify conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization, and that its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. As mutations leading to expression of C-terminally truncated GAS2 have been linked to hearing loss, these findings suggest that the disruption of GAS2-dependent cytoskeletal organisation could underlie auditory dysfunction.
History
DepositionOct 31, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Growth arrest-specific protein 2
B: Tubulin beta chain
C: Detyrosinated tubulin alpha-1B chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,2298
Polymers105,0693
Non-polymers1,1605
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Growth arrest-specific protein 2 / GAS-2


Mass: 8375.974 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Gas2, Gas-2 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P11862
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 47825.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein Detyrosinated tubulin alpha-1B chain


Mass: 48867.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: Q2XVP4

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Non-polymers , 3 types, 5 molecules

#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin
Type: COMPLEX / Entity ID: #2-#3 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 6.8
Details: 80 mM PIPES, pH 6.8, 2 mM MgCl2, 1 mM EGTA, 1mM DTT
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 48.79 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4RELION3CTF correction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
11-25.7182C1
21-25.7182C1
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44922 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT

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