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- PDB-9kbx: CryoEM structure of F-actin bound with GAS2-CH3 domain. -

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Basic information

Entry
Database: PDB / ID: 9kbx
TitleCryoEM structure of F-actin bound with GAS2-CH3 domain.
Components
  • Actin, alpha skeletal muscle
  • Growth arrest-specific protein 2
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 / cytoskeletal motor activator activity / antral ovarian follicle growth / myosin heavy chain binding / tropomyosin binding / actin filament bundle ...initiation of primordial ovarian follicle growth / Caspase-mediated cleavage of cytoskeletal proteins / ovulation / regulation of Notch signaling pathway / basement membrane organization / cytoskeletal motor activator activity / antral ovarian follicle growth / myosin heavy chain binding / tropomyosin binding / actin filament bundle / troponin I binding / filamentous actin / mesenchyme migration / actin filament bundle assembly / skeletal muscle myofibril / striated muscle thin filament / skeletal muscle thin filament assembly / actin monomer binding / skeletal muscle fiber development / stress fiber / titin binding / actin filament polymerization / actin filament / filopodium / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / lamellipodium / regulation of cell shape / cell body / microtubule binding / hydrolase activity / regulation of cell cycle / protein domain specific binding / apoptotic process / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein 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. / 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
ADENOSINE-5'-DIPHOSPHATE / Growth arrest-specific protein 2 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesMus musculus (house mouse)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsAn, J. / Imasaki, T. / Narita, A. / Niwa, S. / Sasaki, R. / Makino, T. / Nitta, R. / Kikkawa, M.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H04762, 21H05248 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121002j002 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: Growth arrest-specific protein 2
H: Actin, alpha skeletal muscle
J: Actin, alpha skeletal muscle
K: Actin, alpha skeletal muscle
L: Actin, alpha skeletal muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,40814
Polymers202,6026
Non-polymers1,8068
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

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


Mass: 18846.986 Da / Num. of mol.: 2
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
Actin, alpha skeletal muscle / Alpha-actin-1


Mass: 41227.035 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)
References: UniProt: P68135, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: Complex of F-actin with GAS2-CH3 domain / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 / Plasmid: pCold Pros2
Buffer solutionpH: 8
Details: 50 mM Tris-HCl, pH 8.0, 200 mM KCl, 2 mM MgCl2, 1 mM EGTA, 4 mM 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
VitrificationCryogen name: ETHANE
Details: The grid was blotted for 4 s at force 10 and plunged into ethane immediately.

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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 magnification: 105000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingElectron dose: 48.93 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 6378
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1RELION4.0bparticle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7MDFFmodel fitting
9RELION4.0binitial Euler assignment
10RELION4.0bfinal Euler assignment
11RELION4.0bclassification
12RELION4.0b3D reconstruction
13PHENIXmodel refinement
CTF correctionDetails: CTF amplitude correction was performed following 3D reconstruction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.55 ° / Axial rise/subunit: 27.7445 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 632290
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 412176 / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingB value: 111 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingAccession code: AF-P11862-F1 / Source name: AlphaFold / Type: in silico model

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