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- EMDB-49903: SPEF1 bound to 13-PF microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-49903
TitleSPEF1 bound to 13-PF microtubule
Map data
Sample
  • Complex: spef1 bound to a 13-pf microtubule
    • Other: SPEF1
    • Other: tubulin alpha 1B
    • Other: tubulin beta 2B
Keywordsmicrotubule / spef1 / cilia / PROTEIN BINDING
Biological speciesHomo sapiens (human) / Bos taurus (domestic cattle)
Methodsubtomogram averaging / cryo EM / Resolution: 8.1 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 25, 2025-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateAug 6, 2025-
Current statusAug 6, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49903.png

Downloads & links

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Map

FileDownload / File: emd_49903.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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Others
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: 0.03
Minimum - Maximum-0.00070174644 - 1.0600921
Average (Standard dev.)0.0017731587 (±0.019718505)
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_49903_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_49903_half_map_2.map
Projections & Slices
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Sample components

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Entire : spef1 bound to a 13-pf microtubule

EntireName: spef1 bound to a 13-pf microtubule
Components
  • Complex: spef1 bound to a 13-pf microtubule
    • Other: SPEF1
    • Other: tubulin alpha 1B
    • Other: tubulin beta 2B

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Supramolecule #1: spef1 bound to a 13-pf microtubule

SupramoleculeName: spef1 bound to a 13-pf microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: SPEF1

MacromoleculeName: SPEF1 / type: other / ID: 1 / Classification: other
Source (natural)Organism: Homo sapiens (human)
SequenceString: MASSVDEEAL HQLYLWVDNI PLSRPKRNLS RDFSDGVLVA EVIKFYFPKM VEMHNYVPAN SLQQKLSNWG HLNRKVLKRL NFSVPDDVMR KIAQCAPGVV ELVLIPLRQR LEERQRRRKQ GAGSLQELAP QDGSGYMDVG VSQKARGEGV PDPQGGGQLS WDRPPAPRPP ...String:
MASSVDEEAL HQLYLWVDNI PLSRPKRNLS RDFSDGVLVA EVIKFYFPKM VEMHNYVPAN SLQQKLSNWG HLNRKVLKRL NFSVPDDVMR KIAQCAPGVV ELVLIPLRQR LEERQRRRKQ GAGSLQELAP QDGSGYMDVG VSQKARGEGV PDPQGGGQLS WDRPPAPRPP AYNRALQGDP SFVLQIAEKE QELLASQETV QVLQMKVRRL EHLLQLKNVR IEDLSRRLQQ AERKQR
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #2: tubulin alpha 1B

MacromoleculeName: tubulin alpha 1B / type: other / ID: 2 / Classification: other
Source (natural)Organism: Bos taurus (domestic cattle)
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHPE QLITGKEDAA NNYARGHYTI GKEIIDLVLD RIRKLADQCT GLQGFLVFHS FGGGTGSGFT SLLMERLSVD YGKKSKLEFS IYPAPQVSTA VVEPYNSILT THTTLEHSDC AFMVDNEAIY DICRRNLDIE RPTYTNLNRL ISQIVSSITA SLRFDGALNV DLTEFQTNLV PYPRIHFPLA TYAPVISAEK AYHEQLSVAE ITNACFEPAN QMVKCDPRHG KYMACCLLYR GDVVPKDVNA AIATIKTKRS IQFVDWCPTG FKVGINYQPP TVVPGGDLAK VQRAVCMLSN TTAIAEAWAR LDHKFDLMYA KRAFVHWYVG EGMEEGEFSE AREDMAALEK DYEEVGVDSV EGEGEEEGEE Y

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Macromolecule #3: tubulin beta 2B

MacromoleculeName: tubulin beta 2B / type: other / ID: 3 / Classification: other
Source (natural)Organism: Bos taurus (domestic cattle)
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVM ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP DRIMNTFSVM PSPKVSDTVV EPYNATLSVH QLVENTDETY CIDNEALYDI CFRTLKLTTP TYGDLNHLVS ATMSGVTTCL RFPGQLNADL RKLAVNMVPF PRLHFFMPGF APLTSRGSQQ YRALTVPELT QQMFDSKNMM AACDPRHGRY LTVAAIFRGR MSMKEVDEQM LNVQNKNSSY FVEWIPNNVK TAVCDIPPRG LKMSATFIGN STAIQELFKR ISEQFTAMFR RKAFLHWYTG EGMDEMEFTE AESNMNDLVS EYQQYQDATA DEQGEFEEEE GEDEA

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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: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 3590
ExtractionNumber tomograms: 58 / Number images used: 12381
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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