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- EMDB-20858: The inner junction complex of Chlamydomonas reinhardtii doublet m... -

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Basic information

Entry
Database: EMDB / ID: EMD-20858
TitleThe inner junction complex of Chlamydomonas reinhardtii doublet microtubule
Map data16-nm repeating unit of Chlamydomonas inner junction
Sample
  • Organelle or cellular component: Inner junction complex of the cilia
    • Protein or peptide: Tubulin beta
    • Protein or peptide: Flagellar associated protein
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: FAP276
    • Protein or peptide: Protein Flattop homolog
    • Protein or peptide: Cilia- and flagella-associated protein 20
    • Protein or peptide: PACRG
    • Protein or peptide: FAP52
Function / homology
Function and homology information


axonemal central pair / axonemal outer doublet / membrane-bounded organelle / 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 / intracellular organelle / motile cilium ...axonemal central pair / axonemal outer doublet / membrane-bounded organelle / 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 / intracellular organelle / motile cilium / microtubule associated complex / cilium assembly / microtubule-based process / Hsp70 protein binding / ciliary basal body / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Hsp90 protein binding / structural constituent of cytoskeleton / microtubule / cytoskeleton / calmodulin binding / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Protein Flattop / Enkurin domain / Calmodulin-binding / Enkurin domain profile. / CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Parkin co-regulated protein / Parkin co-regulated protein / Tubulin-beta mRNA autoregulation signal. ...Protein Flattop / Enkurin domain / Calmodulin-binding / Enkurin domain profile. / CFA20 domain / Cilia- and flagella-associated protein 20/CFAP20DC / CFA20 domain / Parkin co-regulated protein / Parkin co-regulated protein / 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 / Armadillo-type fold / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Cilia- and flagella-associated protein 52 / Flagellar associated protein / Cilia- and flagella-associated protein 20 / Protein Flattop homolog / Uncharacterized protein / Parkin-co-regulated gene product / Tubulin beta-1/beta-2 chain / Tubulin alpha-1 chain
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKhalifa AAZ / Ichikawa M / Bui KH
Funding support Canada, 3 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2016-04954 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-04813 Canada
Canadian Institutes of Health Research (CIHR)PJT-156354 Canada
CitationJournal: Elife / Year: 2020
Title: The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications.
Authors: Ahmad Abdelzaher Zaki Khalifa / Muneyoshi Ichikawa / Daniel Dai / Shintaroh Kubo / Corbin Steven Black / Katya Peri / Thomas S McAlear / Simon Veyron / Shun Kai Yang / Javier Vargas / ...Authors: Ahmad Abdelzaher Zaki Khalifa / Muneyoshi Ichikawa / Daniel Dai / Shintaroh Kubo / Corbin Steven Black / Katya Peri / Thomas S McAlear / Simon Veyron / Shun Kai Yang / Javier Vargas / Susanne Bechstedt / Jean-François Trempe / Khanh Huy Bui /
Abstract: Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate ...Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of and , we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.
History
DepositionOct 23, 2019-
Header (metadata) releaseFeb 5, 2020-
Map releaseFeb 5, 2020-
UpdateFeb 5, 2020-
Current statusFeb 5, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
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  • Surface view with fitted model
  • Atomic models: PDB-6ve7
  • Surface level: 0.04
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ve7
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20858.png

Downloads & links

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Map

FileDownload / File: emd_20858.map.gz / Format: CCP4 / Size: 108.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation16-nm repeating unit of Chlamydomonas inner junction
Voxel sizeX=Y=Z: 0.875 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.078223065 - 0.18503688
Average (Standard dev.)0.0024938365 (±0.020992871)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin21935299
Dimensions234450271
Spacing271234450
CellA: 237.125 Å / B: 204.75 Å / C: 393.75 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8750.8750.875
M x/y/z271234450
origin x/y/z0.0000.0000.000
length x/y/z237.125204.750393.750
α/β/γ90.00090.00090.000
start NX/NY/NZ29921935
NX/NY/NZ271234450
MAP C/R/S321
start NC/NR/NS35219299
NC/NR/NS450234271
D min/max/mean-0.0780.1850.002

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Supplemental data

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Sample components

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Entire : Inner junction complex of the cilia

EntireName: Inner junction complex of the cilia
Components
  • Organelle or cellular component: Inner junction complex of the cilia
    • Protein or peptide: Tubulin beta
    • Protein or peptide: Flagellar associated protein
    • Protein or peptide: Tubulin alpha
    • Protein or peptide: FAP276
    • Protein or peptide: Protein Flattop homolog
    • Protein or peptide: Cilia- and flagella-associated protein 20
    • Protein or peptide: PACRG
    • Protein or peptide: FAP52

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Supramolecule #1: Inner junction complex of the cilia

SupramoleculeName: Inner junction complex of the cilia / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC124 / Organelle: cilia
Molecular weightExperimental: 1 MDa

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Macromolecule #1: Tubulin beta

MacromoleculeName: Tubulin beta / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 49.665809 KDa
SequenceString: MREIVHIQGG QCGNQIGAKF WEVVSDEHGI DPTGTYHGDS DLQLERINVY FNEATGGRYV PRAILMDLEP GTMDSVRSGP YGQIFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAESCDC LQGFQVCHSL GGGTGSGMGT LLISKIREEY P DRMMLTFS ...String:
MREIVHIQGG QCGNQIGAKF WEVVSDEHGI DPTGTYHGDS DLQLERINVY FNEATGGRYV PRAILMDLEP GTMDSVRSGP YGQIFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAESCDC LQGFQVCHSL GGGTGSGMGT LLISKIREEY P DRMMLTFS VVPSPKVSDT VVEPYNATLS VHQLVENADE CMVLDNEALY DICFRTLKLT TPTFGDLNHL ISAVMSGITC CL RFPGQLN ADLRKLAVNL IPFPRLHFFM VGFTPLTSRG SQQYRALTVP ELTQQMWDAK NMMCAADPRH GRYLTASALF RGR MSTKEV DEQMLNVQNK NSSYFVEWIP NNVKSSVCDI PPKGLKMSAT FIGNSTAIQE MFKRVSEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDASAEEEGE FEGEEEEA

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Macromolecule #2: Flagellar associated protein

MacromoleculeName: Flagellar associated protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 27.019803 KDa
SequenceString: MQEESVYALI PQPQEVPQRP AMHTSKFGGK THPAQFDFGQ NKVQPHATMG RPDGANGPAF LHAHEKEPKL PSPGPPSNPK QKIRPPVPA KEEKPTMGLT SNKNFITANA VDVILAKPGK VPQPEFQWTQ KPDYGKVPMY LKRNKDRVAK EKEHFTQYLR M REAPEANA ...String:
MQEESVYALI PQPQEVPQRP AMHTSKFGGK THPAQFDFGQ NKVQPHATMG RPDGANGPAF LHAHEKEPKL PSPGPPSNPK QKIRPPVPA KEEKPTMGLT SNKNFITANA VDVILAKPGK VPQPEFQWTQ KPDYGKVPMY LKRNKDRVAK EKEHFTQYLR M REAPEANA HVSQLSPEDR QQLVRHLKAK WGSVNTAYQG LSLSVDSAVK KGRKEAMERE LAEIERDIRT LERGEVVLVV DD

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Macromolecule #3: Tubulin alpha

MacromoleculeName: Tubulin alpha / type: protein_or_peptide / ID: 3 / Number of copies: 20 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 49.638008 KDa
SequenceString: MREVISIHIG QAGIQVGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRCIFLDL EPTVVDEVRT GTYRQLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLAL DRIRKLADNC TGLQGFLVFN AVGGGTGSGL GSLLLERLSV D YGKKSKLG ...String:
MREVISIHIG QAGIQVGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRCIFLDL EPTVVDEVRT GTYRQLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLAL DRIRKLADNC TGLQGFLVFN AVGGGTGSGL GSLLLERLSV D YGKKSKLG FTVYPSPQVS TAVVEPYNSV LSTHSLLEHT DVAVMLDNEA IYDICRRSLD IERPTYTNLN RLIAQVISSL TA SLRFDGA LNVDITEFQT NLVPYPRIHF MLSSYAPIIS AEKAYHEQLS VAEITNAAFE PASMMVKCDP RHGKYMACCL MYR GDVVPK DVNASVATIK TKRTIQFVDW CPTGFKCGIN YQPPTVVPGG DLAKVQRAVC MISNSTAIGE IFSRLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDL AALEKDFEEV GAESAEGAGE GEGEEY

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Macromolecule #4: FAP276

MacromoleculeName: FAP276 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 10.073385 KDa
SequenceString:
MDLKQQVKNY TMTIRNTRPP TMIKEQDKSE FSHFRALQVL ANGDEVPYEA TLRNVIHDGA RQPKLPPRQT QKHPGYIRNE SGGFFTS

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Macromolecule #5: Protein Flattop homolog

MacromoleculeName: Protein Flattop homolog / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 15.435397 KDa
SequenceString:
MSRSYPGEQV EHAFNSKRLK NWEVPAVDKS QAISTSTGTR FGTLQPRSGR TQFIVDDNGH LKSGVPKLEK SAFNFTQTTP VFMDSAPRW PKENPTWPKN MKATMGYKGI QSNYLPTNTV TLKAVEVPGT TERNFNFM

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Macromolecule #6: Cilia- and flagella-associated protein 20

MacromoleculeName: Cilia- and flagella-associated protein 20 / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 22.193566 KDa
SequenceString:
MFKNAFQSGF LSVLYSIGSK PLEIWDKQVS NGHIKRITDA DIQSSVLEIM GQNVSTTYIT CPADPNKTLG IKLPFLVLII KNLNKYFSF EVQVLDDKNV RRRFRASNYQ STTRVKPFIC TMPMRLDSGW NQIQFNLSDF TRRAYGTNYI ETLRVQVHAN C RIRRIYFS DRLYSEEELP AEFKLFLPIQ KS

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Macromolecule #7: PACRG

MacromoleculeName: PACRG / type: protein_or_peptide / ID: 7 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 34.215148 KDa
SequenceString: MNGDVAGSLF TSTYRNVKLA GKAPPAANLS GTGSCFDTTS LSPARAGAHK ALDVQKDELP VWSKSTLSYK YPAGRPNPTG FLKKGDGEM IKTKTGGFEE RKPSPPQAGA YKRRENPPNT AFRRFYERGD LPIAVDHRGS KNMIAWKVDI EKLDYHHYLP I FFDGIRET ...String:
MNGDVAGSLF TSTYRNVKLA GKAPPAANLS GTGSCFDTTS LSPARAGAHK ALDVQKDELP VWSKSTLSYK YPAGRPNPTG FLKKGDGEM IKTKTGGFEE RKPSPPQAGA YKRRENPPNT AFRRFYERGD LPIAVDHRGS KNMIAWKVDI EKLDYHHYLP I FFDGIRET QEPYRFLAVK GVEDMLRVGG SKILPVIPQL IIPIKTALNT RDHSVMCITL QLLQKLVLSA DLVGEALVPY YR QILPIFN LYKNKNKNLG DGIDYGQRNY DCLGELIADT LALFEQKGGD DAFINIKYMV PTYESSVNYA

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Macromolecule #8: FAP52

MacromoleculeName: FAP52 / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 68.546508 KDa
SequenceString: MAEPLVLNSV IGFGGAIENG LIAHPDGRTI IYPLGSTIVL RDRADPRSQE FLQGHSDKVS CLALSRSGRY LASGQITYMG FTADIIIWD LESKQLIHRM ALHKVKVQAL DFSCDERFLA SLGGQDDNAL VLWDVASGNA ICGSPCNTNF TNCVKFFNNS P DKLITAGN ...String:
MAEPLVLNSV IGFGGAIENG LIAHPDGRTI IYPLGSTIVL RDRADPRSQE FLQGHSDKVS CLALSRSGRY LASGQITYMG FTADIIIWD LESKQLIHRM ALHKVKVQAL DFSCDERFLA SLGGQDDNAL VLWDVASGNA ICGSPCNTNF TNCVKFFNNS P DKLITAGN FNMNVWTYDA GNNKLRPTDA TLGTLKRVFK SVVVDANDEY AYCGTTTGDV LQIALERVLF KNTGPAKGNV QM GVTATCE VPTGDILVGG GDGSLQVLRT VPEPSSTNPK LLRKMPALAG TKVEGAITSI ALADMNARGF TFFVGTAMCN IYK VTYEPA TSRLKEELVQ TAHNDKINGM AFPNEYSEVF ATCGTGFIRL WHLTTCRELL RIAVPNLECF CIAFTTDGSA ILSG WSDGK IRAFGPQSGK IIFTINDAHQ KAVTAIASTA DSSRILSGGE EGMVRVWRIG RTSQTLEASM KDHKGPVNCI RIKGS GDEC VSASSDGSCI LWDLHTFKRR TSLFANTFFK SVVYHPDESQ LVTAGTDRKV TYWDAYDGNA IRIIDGSDLD EVNALA VDR DGEALVSGGG DKLVKLWGYD EGHCYFVGVA HSGAITAVGV TPDKQRIVSV GTEGGIFIWD YQRPQTLADI

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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

Concentration4 mg/mL
BufferpH: 7.4
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.8 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-7 / Number real images: 9528 / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 270713
CTF correctionSoftware - Name: RELION (ver. 3.0)
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER (ver. 19.02)
Final 3D classificationNumber classes: 1 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 270713
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6ve7:
The inner junction complex of Chlamydomonas reinhardtii doublet microtubule

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