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- EMDB-62233: CryoEM structure of F-actin bound with GAS2-CH3 domain. -

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Basic information

Entry
Database: EMDB / ID: EMD-62233
TitleCryoEM structure of F-actin bound with GAS2-CH3 domain.
Map datasharpened map
Sample
  • Complex: Complex of F-actin with GAS2-CH3 domain
    • Protein or peptide: Growth arrest-specific protein 2
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsCytoskeleton / STRUCTURAL PROTEIN
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 / regulation of cell shape / lamellipodium / cell body / microtubule binding / regulation of cell cycle / hydrolase activity / 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
Growth arrest-specific protein 2 / Actin, alpha skeletal muscle
Similarity search - Component
Biological speciesMus musculus (house mouse) / Oryctolagus cuniculus (rabbit)
Methodhelical 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, 2 items
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, 2024-
Header (metadata) releaseSep 10, 2025-
Map releaseSep 10, 2025-
UpdateSep 10, 2025-
Current statusSep 10, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62233.png

Downloads & links

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Map

FileDownload / File: emd_62233.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å		0.83 Å/pix.
x 384 pix.
= 318.72 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0112
Minimum - Maximum-0.09346453 - 0.20898336
Average (Standard dev.)0.00007610787 (±0.0032709034)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62233_msk_1.map
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Additional map: raw map

Fileemd_62233_additional_1.map
Annotationraw map
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Half map: #2

Fileemd_62233_half_map_1.map
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Half map: #1

Fileemd_62233_half_map_2.map
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Sample components

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Entire : Complex of F-actin with GAS2-CH3 domain

EntireName: Complex of F-actin with GAS2-CH3 domain
Components
  • Complex: Complex of F-actin with GAS2-CH3 domain
    • Protein or peptide: Growth arrest-specific protein 2
    • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of F-actin with GAS2-CH3 domain

SupramoleculeName: Complex of F-actin with GAS2-CH3 domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Growth arrest-specific protein 2

MacromoleculeName: Growth arrest-specific protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 18.846986 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MMCTALSPKV RSGPGLSDMH QYSQWLASRH EANLLPMKED LALWLTNLLG KEITAETFME KLDNGALLCQ LAATVQEKFK ESMDANKPA KTLPLKKIPC KASAPSGSFF ARDNTANFLS WCRDLGVDET CLFESEGLVL HKQPREVCLC LLELGRIAAR Y GVEPPGLI KL

UniProtKB: Growth arrest-specific protein 2

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Macromolecule #2: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.227035 KDa
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIEHG IITNWDDMEK IWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVTHNV P IYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIEHG IITNWDDMEK IWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVTHNV P IYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSYEL PD GQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITALA PST MKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKC

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
Details: 50 mM Tris-HCl, pH 8.0, 200 mM KCl, 2 mM MgCl2, 1 mM EGTA, 4 mM DTT.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 6378 / Average electron dose: 48.93 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 27.7445 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.55 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0b) / Number images used: 412176
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Details: CTF amplitude correction was performed following 3D reconstruction.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Segment selectionNumber selected: 632290 / Software - Name: RELION (ver. 4.0b)
Startup modelType of model: OTHER / Details: AlphaFold AF-P11862-F1
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 4.0b)

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Atomic model buiding 1

Initial modelPDB ID:

2-f1


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 111
Output model

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

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