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- EMDB-49760: SPEF1 bound to 14-pf microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-49760
TitleSPEF1 bound to 14-pf microtubule
Map data
Sample
  • Complex: 14-protofilament microtubule bound by SPEF1 CH-domain
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Sperm flagellar protein 1,G protein/GFP fusion protein
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL
Keywordsmicrotubules / spef1 / seam / PROTEIN BINDING
Function / homology
Function and homology information


axonemal central apparatus / axonemal central apparatus assembly / regulation of Wnt signaling pathway, planar cell polarity pathway / 9+2 motile cilium / cilium movement / ciliary tip / negative regulation of microtubule depolymerization / filopodium assembly / microtubule bundle formation / lamellipodium assembly ...axonemal central apparatus / axonemal central apparatus assembly / regulation of Wnt signaling pathway, planar cell polarity pathway / 9+2 motile cilium / cilium movement / ciliary tip / negative regulation of microtubule depolymerization / filopodium assembly / microtubule bundle formation / lamellipodium assembly / regulation of cytoskeleton organization / microvillus / axoneme / host cell membrane / microtubule-based process / cytoplasmic microtubule / stress fiber / cellular response to interleukin-4 / bioluminescence / generation of precursor metabolites and energy / filopodium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / cell migration / mitotic cell cycle / lamellipodium / double-stranded RNA binding / actin binding / microtubule cytoskeleton / basolateral plasma membrane / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cilium / apical plasma membrane / symbiont entry into host cell / GTPase activity / viral envelope / ubiquitin protein ligase binding / GTP binding / virion attachment to host cell / virion membrane / metal ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
: / CH-like domain in sperm protein / CH-like domain in sperm protein / : / Rhabdovirus spike glycoprotein G central domain / Rhabdovirus glycoprotein / Rhabdovirus spike glycoprotein fusion domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. ...: / CH-like domain in sperm protein / CH-like domain in sperm protein / : / Rhabdovirus spike glycoprotein G central domain / Rhabdovirus glycoprotein / Rhabdovirus spike glycoprotein fusion 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 / Green fluorescent protein, GFP / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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 / G protein/GFP fusion protein / Tubulin alpha-1B chain / Sperm flagellar protein 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Recombinant vesicular stomatitis Indiana virus rVSV-G/GFP / Bos taurus (domestic cattle)
Methodsubtomogram averaging / cryo EM / Resolution: 7.1 Å
AuthorsLegal T / Bui KH
Funding support Canada, 2 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-156354 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2022-04774 Canada
CitationJournal: Curr Biol / Year: 2025
Title: Structure of the ciliary tip central pair reveals the unique role of the microtubule-seam binding protein SPEF1.
Authors: Thibault Legal / Ewa Joachimiak / Mireya Parra / Wang Peng / Amanda Tam / Corbin Black / Mayukh Guha / Chau Anh Nguyen / Avrin Ghanaeian / Melissa Valente-Paterno / Gary Brouhard / Jacek ...Authors: Thibault Legal / Ewa Joachimiak / Mireya Parra / Wang Peng / Amanda Tam / Corbin Black / Mayukh Guha / Chau Anh Nguyen / Avrin Ghanaeian / Melissa Valente-Paterno / Gary Brouhard / Jacek Gaertig / Dorota Wloga / Khanh Huy Bui /
Abstract: Motile cilia are unique organelles with the ability to move autonomously. The force generated by beating cilia propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet ...Motile cilia are unique organelles with the ability to move autonomously. The force generated by beating cilia propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet microtubules and a central pair (CP) with a distinct structure at the tip. In this study, we present a high-resolution structure of the CP in the ciliary tip of the ciliate Tetrahymena thermophila and identify several tip proteins that bind and form unique patterns on both microtubules of the tip CP. Two of those proteins that contain tubulin polymerization-promoting protein (TPPP)-like domains, TLP1 and TLP2, bind to high curvature regions of the microtubule. TLP2, which contains two TPPP-like domains, is an unusually long protein that wraps laterally around half a microtubule and forms the bridge between the two microtubules. Moreover, we found that the conserved protein SPEF1 binds to both microtubule seams and crosslinked the two microtubules. In vitro, human SPEF1 binds to the microtubule seam as visualized by cryoelectron tomography and subtomogram averaging. Single-molecule microtubule dynamics assays indicate that SPEF1 stabilizes microtubules in vitro. Together, these data show that the proteins in the tip CP maintain stable microtubule structures and play important roles in maintaining the integrity of the axoneme.
History
DepositionMar 18, 2025-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49760.png

Downloads & links

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Map

FileDownload / File: emd_49760.map.gz / Format: CCP4 / Size: 206 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 378 pix.
= 801.36 Å		2.12 Å/pix.
x 378 pix.
= 801.36 Å		2.12 Å/pix.
x 378 pix.
= 801.36 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0398
Minimum - Maximum-0.0004562203 - 1.363461
Average (Standard dev.)0.004955031 (±0.041669276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions378378378
Spacing378378378
CellA=B=C: 801.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49760_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_49760_half_map_2.map
Projections & Slices
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Sample components

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Entire : 14-protofilament microtubule bound by SPEF1 CH-domain

EntireName: 14-protofilament microtubule bound by SPEF1 CH-domain
Components
  • Complex: 14-protofilament microtubule bound by SPEF1 CH-domain
    • Protein or peptide: Tubulin alpha-1B chain
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Sperm flagellar protein 1,G protein/GFP fusion protein
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOL

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Supramolecule #1: 14-protofilament microtubule bound by SPEF1 CH-domain

SupramoleculeName: 14-protofilament microtubule bound by SPEF1 CH-domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#3, #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Sperm flagellar protein 1,G protein/GFP fusion protein

MacromoleculeName: Sperm flagellar protein 1,G protein/GFP fusion protein
type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Recombinant vesicular stomatitis Indiana virus rVSV-G/GFP
Molecular weightTheoretical: 44.324262 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: PTMGSSGSSG MASSVDEEAL HQLYLWVDNI PLSRPKRNLS RDFSDGVLVA EVIKFYFPKM VEMHNYVPAN SLQQKLSNWG HLNRKVLKR LNFSVPDDVM RKIAQCAPGV VELVLIPLRQ RLEERQRRRK QGAGSLSSPQ QMVSKGEELF TGVVPILVEL D GDVNGHKF ...String:
PTMGSSGSSG MASSVDEEAL HQLYLWVDNI PLSRPKRNLS RDFSDGVLVA EVIKFYFPKM VEMHNYVPAN SLQQKLSNWG HLNRKVLKR LNFSVPDDVM RKIAQCAPGV VELVLIPLRQ RLEERQRRRK QGAGSLSSPQ QMVSKGEELF TGVVPILVEL D GDVNGHKF SVSGEGEGDA TYGKLTLKFI CTTGKLPVPW PTLVTTLTYG VQCFSRYPDH MKQHDFFKSA MPEGYVQERT IF FKDDGNY KTRAEVKFEG DTLVNRIELK GIDFKEDGNI LGHKLEYNYN SHNVYIMADK QKNGIKVNFK IRHNIEDGSV QLA DHYQQN TPIGDGPVLL PDNHYLSTQS ALSKDPNEKR DHMVLLEFVT AAGITLGMDE LYKGSAAAAW SHPQFEK

UniProtKB: Sperm flagellar protein 1, G protein/GFP fusion protein

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Macromolecule #2: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 2 / Number of copies: 42 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 50.204445 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 GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #3: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 3 / Number of copies: 42 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 49.983824 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VMPSPKVSDT VVEPYNATLS VHQLVENTDE TYSIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDSK NMMAACDPRH GRYLTVAAIF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEEGEDEA

UniProtKB: Tubulin beta chain

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 42 / 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 #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 42 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 42 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Macromolecule #7: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 7 / Number of copies: 42 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 4.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 9625
ExtractionNumber tomograms: 58 / Number images used: 20342
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Final angle assignmentType: MAXIMUM LIKELIHOOD

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