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- PDB-9ntm: SPEF1 bound to 14-pf microtubule -

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Basic information

Entry
Database: PDB / ID: 9ntm
TitleSPEF1 bound to 14-pf microtubule
Components
  • Sperm flagellar protein 1,G protein/GFP fusion protein
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsPROTEIN BINDING / microtubules / spef1 / seam
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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / 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)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.1 Å
AuthorsLegal, T. / Bui, K.H.
Funding support Canada, 2items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1A: Sperm flagellar protein 1,G protein/GFP fusion protein
1B: Sperm flagellar protein 1,G protein/GFP fusion protein
1C: Sperm flagellar protein 1,G protein/GFP fusion protein
1D: Sperm flagellar protein 1,G protein/GFP fusion protein
1E: Sperm flagellar protein 1,G protein/GFP fusion protein
AA: Tubulin alpha-1B chain
AB: Tubulin beta chain
AC: Tubulin alpha-1B chain
AD: Tubulin beta chain
AE: Tubulin alpha-1B chain
AF: Tubulin beta chain
BA: Tubulin alpha-1B chain
BB: Tubulin beta chain
BC: Tubulin alpha-1B chain
BD: Tubulin beta chain
BE: Tubulin alpha-1B chain
BF: Tubulin beta chain
CA: Tubulin alpha-1B chain
CB: Tubulin beta chain
CC: Tubulin alpha-1B chain
CD: Tubulin beta chain
CE: Tubulin alpha-1B chain
CF: Tubulin beta chain
DA: Tubulin alpha-1B chain
DB: Tubulin beta chain
DC: Tubulin alpha-1B chain
DD: Tubulin beta chain
DE: Tubulin alpha-1B chain
DF: Tubulin beta chain
EA: Tubulin alpha-1B chain
EB: Tubulin beta chain
EC: Tubulin alpha-1B chain
ED: Tubulin beta chain
EE: Tubulin alpha-1B chain
EF: Tubulin beta chain
FA: Tubulin alpha-1B chain
FB: Tubulin beta chain
FC: Tubulin alpha-1B chain
FD: Tubulin beta chain
FE: Tubulin alpha-1B chain
FF: Tubulin beta chain
GA: Tubulin alpha-1B chain
GB: Tubulin beta chain
GC: Tubulin alpha-1B chain
GD: Tubulin beta chain
GE: Tubulin alpha-1B chain
GF: Tubulin beta chain
HA: Tubulin alpha-1B chain
HB: Tubulin beta chain
HC: Tubulin alpha-1B chain
HD: Tubulin beta chain
HE: Tubulin alpha-1B chain
HF: Tubulin beta chain
IA: Tubulin alpha-1B chain
IB: Tubulin beta chain
IC: Tubulin alpha-1B chain
ID: Tubulin beta chain
IE: Tubulin alpha-1B chain
IF: Tubulin beta chain
JA: Tubulin alpha-1B chain
JB: Tubulin beta chain
JC: Tubulin alpha-1B chain
JD: Tubulin beta chain
JE: Tubulin alpha-1B chain
JF: Tubulin beta chain
KA: Tubulin alpha-1B chain
KB: Tubulin beta chain
KC: Tubulin alpha-1B chain
KD: Tubulin beta chain
KE: Tubulin alpha-1B chain
KF: Tubulin beta chain
LA: Tubulin alpha-1B chain
LB: Tubulin beta chain
LC: Tubulin alpha-1B chain
LD: Tubulin beta chain
LE: Tubulin alpha-1B chain
LF: Tubulin beta chain
MA: Tubulin alpha-1B chain
MB: Tubulin beta chain
MC: Tubulin alpha-1B chain
MD: Tubulin beta chain
ME: Tubulin alpha-1B chain
MF: Tubulin beta chain
NA: Tubulin alpha-1B chain
NB: Tubulin beta chain
NC: Tubulin alpha-1B chain
ND: Tubulin beta chain
NE: Tubulin alpha-1B chain
NF: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)4,507,001257
Polymers4,429,52989
Non-polymers77,473168
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 89 molecules 1A1B1C1D1EAAACAEBABCBECACCCEDADCDEEAECEEFAFCFEGAGCGEHAHCHEIA...

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


Mass: 44324.262 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Recombinant vesicular stomatitis Indiana virus rVSV-G/GFP
Gene: SPEF1, C20orf28, G, GFP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9Y4P9, UniProt: B7UCZ6
#2: Protein ...
Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 42 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P81947
#3: Protein ...
Tubulin beta chain


Mass: 49983.824 Da / Num. of mol.: 42 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: A0A4W2DT89

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Non-polymers , 4 types, 168 molecules

#4: Chemical...
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: Mg
#6: Chemical...
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical...
ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 42 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: 14-protofilament microtubule bound by SPEF1 CH-domain / Type: COMPLEX / Entity ID: #2-#3, #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
21Bos taurus (domestic cattle)9913
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 4000 nm / Nominal defocus min: 2000 nm
Image recordingElectron dose: 4 e/Å2 / Avg electron dose per subtomogram: 164 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9625 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 58 / Num. of volumes extracted: 20342

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