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- EMDB-62232: CryoEM structure of microtubule bound with GAS2-GAR domain. -

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Basic information

Entry
Database: EMDB / ID: EMD-62232
TitleCryoEM structure of microtubule bound with GAS2-GAR domain.
Map dataMiRP GAS2-GAR-MT raw map
Sample
  • Complex: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Detyrosinated tubulin alpha-1B chain
  • Protein or peptide: Growth arrest-specific protein 2
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • 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 / 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. / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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
Tubulin beta chain / Growth arrest-specific protein 2 / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesMus musculus (house mouse) / Sus scrofa (pig)
Methodhelical 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, 1 items
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, 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_62232.png

Downloads & links

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Map

FileDownload / File: emd_62232.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMiRP GAS2-GAR-MT raw map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 540 pix.
= 448.2 Å		0.83 Å/pix.
x 540 pix.
= 448.2 Å		0.83 Å/pix.
x 540 pix.
= 448.2 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: 0.005
Minimum - Maximum-0.027385049 - 0.05814774
Average (Standard dev.)0.000109891815 (±0.003156251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 448.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62232_msk_1.map
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Additional map: CryoSPARC GAS2-GAR-MT raw map

Fileemd_62232_additional_1.map
AnnotationCryoSPARC GAS2-GAR-MT raw map
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Additional map: MiRP GAS2-GAR-MT masked map

Fileemd_62232_additional_2.map
AnnotationMiRP GAS2-GAR-MT masked map
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Additional map: GAS2-FL-MT map

Fileemd_62232_additional_3.map
AnnotationGAS2-FL-MT map
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Half map: Half map1 for GAS2-GAR-MT

Fileemd_62232_half_map_1.map
AnnotationHalf map1 for GAS2-GAR-MT
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Half map: half map2 for GAS2-GAR-MT

Fileemd_62232_half_map_2.map
Annotationhalf map2 for GAS2-GAR-MT
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Sample components

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Entire : Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin

EntireName: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin
Components
  • Complex: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Detyrosinated tubulin alpha-1B chain
  • Protein or peptide: Growth arrest-specific protein 2
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin

SupramoleculeName: Ternary complex of GAS2 GAR domain with alpha-tubulin and beta-tubulin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3
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: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 8.375974 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
LLDDAVKRIS EDPPCKCPTK FCVERLSQGR YRVGEKILFI RMLHNKHVMV RVGGGWETFA GYLLKHDPCR ML

UniProtKB: Growth arrest-specific protein 2

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 47.825859 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY Q

UniProtKB: Tubulin beta chain

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Macromolecule #3: Detyrosinated tubulin alpha-1B chain

MacromoleculeName: Detyrosinated tubulin alpha-1B chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 48.867195 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDS

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / 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: 6.8
Details: 80 mM PIPES, pH 6.8, 2 mM MgCl2, 1 mM EGTA, 1mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.79 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: 1.8 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 82.0 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.71000 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 44922
CTF correctionSoftware - Name: RELION (ver. 3.0) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1v5r

Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

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

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