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- EMDB-34836: Complex of GMPCPP microtubule, FAP20 and tubulin (in absence of GTP) -

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Basic information

Entry
Database: EMDB / ID: EMD-34836
TitleComplex of GMPCPP microtubule, FAP20 and tubulin (in absence of GTP)
Map dataC1 reconstruction of microtubule:FAP20:tubulin complex
Sample
  • Complex: Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP
    • Complex: tublin
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Cilia- and flagella-associated protein 20
      • Protein or peptide: Cilia- and flagella-associated protein 20
Keywordsdoublet microtubule / cilia- and flagella- associated protein 20 (FAP20) / inner junction protein / cilia / STRUCTURAL PROTEIN
Function / homology
Function and homology information


axonemal central pair / axonemal doublet microtubule / positive regulation of cilium-dependent cell motility / regulation of cilium beat frequency involved in ciliary motility / establishment of protein localization to organelle / axoneme assembly / axonemal microtubule / embryonic brain development / motile cilium / positive regulation of axon guidance ...axonemal central pair / axonemal doublet microtubule / positive regulation of cilium-dependent cell motility / regulation of cilium beat frequency involved in ciliary motility / establishment of protein localization to organelle / axoneme assembly / axonemal microtubule / embryonic brain development / motile cilium / positive regulation of axon guidance / microtubule associated complex / intercellular bridge / microtubule-based process / cilium assembly / ciliary basal body / neuron migration / Schaffer collateral - CA1 synapse / modulation of chemical synaptic transmission / structural constituent of cytoskeleton / mitotic spindle / cerebral cortex development / microtubule / hydrolase activity / protein heterodimerization activity / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain ...CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 alpha chain / Tubulin beta chain / Cilia- and flagella-associated protein 20
Similarity search - Component
Biological speciesCapra hircus (goat) / Chlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsBangera M / Sirajuddin M
Funding support India, European Union, 6 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/14/2/501533 India
Department of Science & Technology (DST, India)CEFIPRA (5703-1) India
Science and Engineering Research Board (SERB)CRG/2019/003246 India
Department of Biotechnology (DBT, India)BT/PR40389/COT/142/6/2020 India
European Molecular Biology Organization (EMBO)Young Investigator ProgrammeEuropean Union
Science and Engineering Research Board (SERB)PDF/2016/002682 India
CitationJournal: Structure / Year: 2023
Title: Doublet microtubule inner junction protein FAP20 recruits tubulin to the microtubule lattice.
Authors: Mamata Bangera / Archita Dungdung / Sujana Prabhu / Minhajuddin Sirajuddin /
Abstract: Doublet microtubules of eukaryotic cilia and flagella are made up of a complete A- and an incomplete B-tubule that are fused together. Of the two fusion points, the outer junction is made of ...Doublet microtubules of eukaryotic cilia and flagella are made up of a complete A- and an incomplete B-tubule that are fused together. Of the two fusion points, the outer junction is made of tripartite tubulin connections, while the inner junction contains non-tubulin elements. The latter includes flagellar-associated protein 20 (FAP20) and Parkin co-regulated gene protein (PACRG) that together link the A- and B-tubule at the inner junction. While structures of doublet microtubules reveal molecular details, their assembly is poorly understood. In this study, we purified recombinant FAP20 and characterized its effects on microtubule dynamics. We use in vitro reconstitution and cryo-electron microscopy to show that FAP20 recruits free tubulin to the existing microtubule lattice. Our cryo-electron microscopy reconstruction of microtubule:FAP20:tubulin complex reveals the mode of tubulin recruitment by FAP20 onto microtubules, providing insights into assembly steps of B-tubule closure during doublet microtubule formation.
History
DepositionNov 22, 2022-
Header (metadata) releaseOct 4, 2023-
Map releaseOct 4, 2023-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34836.png

Downloads & links

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Map

FileDownload / File: emd_34836.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC1 reconstruction of microtubule:FAP20:tubulin complex
Voxel sizeX=Y=Z: 1.78 Å
Density
Contour LevelBy AUTHOR: 0.022
Minimum - Maximum-0.052052066 - 0.07897297
Average (Standard dev.)0.0014676097 (±0.0131524475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 783.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 1 for C1 reconstruction of microtubule:FAP20:tubulin complex

Fileemd_34836_half_map_1.map
AnnotationHalf map 1 for C1 reconstruction of microtubule:FAP20:tubulin complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 for C1 reconstruction of microtubule:FAP20:tubulin complex

Fileemd_34836_half_map_2.map
AnnotationHalf map 2 for C1 reconstruction of microtubule:FAP20:tubulin complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP

EntireName: Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP
Components
  • Complex: Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP
    • Complex: tublin
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta chain
    • Complex: Cilia- and flagella-associated protein 20
      • Protein or peptide: Cilia- and flagella-associated protein 20

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Supramolecule #1: Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP

SupramoleculeName: Complex of GMPCPP microtubule with FAP20 and tubulin in absence of GTP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: tublin

SupramoleculeName: tublin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Capra hircus (goat)

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Supramolecule #3: Cilia- and flagella-associated protein 20

SupramoleculeName: Cilia- and flagella-associated protein 20 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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Macromolecule #1: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Capra hircus (goat)
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

UniProtKB: Tubulin alpha chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Capra hircus (goat)
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

UniProtKB: Tubulin beta chain

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Macromolecule #3: Cilia- and flagella-associated protein 20

MacromoleculeName: Cilia- and flagella-associated protein 20 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFKNAFQSGF LSVLYSIGSK PLEIWDKQVS NGHIKRITDA DIQSSVLEIM GQNVSTTYIT CPADPNKTLG IKLPFLVLII KNLNKYFSFE VQVLDDKNVR RRFRASNYQS TTRVKPFICT MPMRLDSGWN QIQFNLSDFT RRAYGTNYIE TLRVQVHANC RIRRIYFSDR ...String:
MFKNAFQSGF LSVLYSIGSK PLEIWDKQVS NGHIKRITDA DIQSSVLEIM GQNVSTTYIT CPADPNKTLG IKLPFLVLII KNLNKYFSFE VQVLDDKNVR RRFRASNYQS TTRVKPFICT MPMRLDSGWN QIQFNLSDFT RRAYGTNYIE TLRVQVHANC RIRRIYFSDR LYSEEELPAE FKLFLPIQKS LEVLFQGPGS HHHHHHHHH

UniProtKB: Cilia- and flagella-associated protein 20

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: BRB80 buffer : 80mM PIPES, 2mM MgCl2, 1mM EGTA supplemented with 1mM GMPCPP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.025 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: GMPCPP MT seeds in warm BRB80 was applied to the grid followed by second application of pre-warmed solution containing FAP20 and tubulin. After an incubation time of 1 minute, grid was ...Details: GMPCPP MT seeds in warm BRB80 was applied to the grid followed by second application of pre-warmed solution containing FAP20 and tubulin. After an incubation time of 1 minute, grid was blotted for 3 seconds before plunging into liquid ethane..
DetailsGoat brain tubulin was polymerized with 1mM GMPCPP at 310K for 2 hours, spun on warm 50 percent BRB80 sucrose cushion and resuspended in warm BRB80 buffer.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1467 / Average exposure time: 2.0 sec. / Average electron dose: 39.55 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 24621
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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