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- EMDB-41090: Composite map of TTLL6 bound to unmodified human microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-41090
TitleComposite map of TTLL6 bound to unmodified human microtubule
Map dataComposite map of TTLL6 bound to unmodified human microtubules.
Sample
  • Complex: TTLL6 bound to unmodified human microtubule
    • Complex: alpha1B and betaI/betaIVb microtubule
      • Other: Tubulin alpha-1B chain
      • Other: Tubulin beta chain
    • Complex: Tubulin polyglutamylase TTLL6 in complex with unmodified human microtubule
      • Other: Tubulin polyglutamylase TTLL6
Keywordstubulin post-translational modifications / microtubules / TTLL / LIGASE
Function / homology
Function and homology information


positive regulation of cilium movement / protein-glutamic acid ligase activity / tubulin-glutamic acid ligase activity / Carboxyterminal post-translational modifications of tubulin / protein polyglutamylation / regulation of cilium beat frequency involved in ciliary motility / 9+0 non-motile cilium / microtubule severing / Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases) / odontoblast differentiation ...positive regulation of cilium movement / protein-glutamic acid ligase activity / tubulin-glutamic acid ligase activity / Carboxyterminal post-translational modifications of tubulin / protein polyglutamylation / regulation of cilium beat frequency involved in ciliary motility / 9+0 non-motile cilium / microtubule severing / Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases) / odontoblast differentiation / Post-chaperonin tubulin folding pathway / Carboxyterminal post-translational modifications of tubulin / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / cytoskeleton-dependent intracellular transport / Formation of tubulin folding intermediates by CCT/TriC / Gap junction assembly / COPI-independent Golgi-to-ER retrograde traffic / natural killer cell mediated cytotoxicity / microtubule bundle formation / Assembly and cell surface presentation of NMDA receptors / Kinesins / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / regulation of synapse organization / nuclear envelope lumen / cytoplasmic microtubule / Recycling pathway of L1 / MHC class I protein binding / RHOH GTPase cycle / RHO GTPases activate IQGAPs / spindle assembly / cellular response to interleukin-4 / microtubule-based process / Hedgehog 'off' state / COPI-mediated anterograde transport / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / MHC class II antigen presentation / tubulin binding / AURKA Activation by TPX2 / ciliary basal body / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / PKR-mediated signaling / cilium / mitotic spindle / structural constituent of cytoskeleton / microtubule cytoskeleton organization / Aggrephagy / cytoplasmic ribonucleoprotein granule / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / Regulation of PLK1 Activity at G2/M Transition / azurophil granule lumen / double-stranded RNA binding / mitotic cell cycle / cell body / microtubule / Potential therapeutics for SARS / cytoskeleton / membrane raft / protein domain specific binding / cell division / GTPase activity / ubiquitin protein ligase binding / Neutrophil degranulation / protein-containing complex binding / GTP binding / structural molecule activity / protein-containing complex / extracellular exosome / extracellular region / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Tubulin-tyrosine ligase/Tubulin polyglutamylase / Tubulin-tyrosine ligase family / TTL domain profile. / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain ...Tubulin-tyrosine ligase/Tubulin polyglutamylase / Tubulin-tyrosine ligase family / TTL domain profile. / 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
Similarity search - Domain/homology
Tubulin polyglutamylase TTLL6 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsMahalingan KK / Grotjahn D / Li Y / Lander GC / Zehr EA / Roll-Mecak A
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)ZO1 NS003163 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Structural basis for α-tubulin-specific and modification state-dependent glutamylation.
Authors: Kishore K Mahalingan / Danielle A Grotjahn / Yan Li / Gabriel C Lander / Elena A Zehr / Antonina Roll-Mecak /
Abstract: Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and β-tubulin. ...Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and β-tubulin. How TTLLs specialize for specific substrate recognition and ultimately modification-pattern generation is largely unknown. TTLL6, a glutamylase implicated in ciliopathies, preferentially modifies tubulin α-tails in microtubules. Cryo-electron microscopy, kinetic analysis and single-molecule biochemistry reveal an unprecedented quadrivalent recognition that ensures simultaneous readout of microtubule geometry and posttranslational modification status. By binding to a β-tubulin subunit, TTLL6 modifies the α-tail of the longitudinally adjacent tubulin dimer. Spanning two tubulin dimers along and across protofilaments (PFs) ensures fidelity of recognition of both the α-tail and the microtubule. Moreover, TTLL6 reads out and is stimulated by glutamylation of the β-tail of the laterally adjacent tubulin dimer, mediating crosstalk between α-tail and β-tail. This positive feedback loop can generate localized microtubule glutamylation patterns. Our work uncovers general principles that generate tubulin chemical and topographic complexity.
History
DepositionJun 20, 2023-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41090.png

Downloads & links

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Map

FileDownload / File: emd_41090.map.gz / Format: CCP4 / Size: 699 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map of TTLL6 bound to unmodified human microtubules.
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 2.23
Minimum - Maximum-32.464689999999997 - 64.712429999999998
Average (Standard dev.)0.014137393 (±0.528074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions568568568
Spacing568568568
CellA=B=C: 653.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Second half-map of TTLL6 bound to unmodified human microtubules.

Fileemd_41090_half_map_1.map
AnnotationSecond half-map of TTLL6 bound to unmodified human microtubules.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First half-map of TTLL6 bound to unmodified human microtubules.

Fileemd_41090_half_map_2.map
AnnotationFirst half-map of TTLL6 bound to unmodified human microtubules.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TTLL6 bound to unmodified human microtubule

EntireName: TTLL6 bound to unmodified human microtubule
Components
  • Complex: TTLL6 bound to unmodified human microtubule
    • Complex: alpha1B and betaI/betaIVb microtubule
      • Other: Tubulin alpha-1B chain
      • Other: Tubulin beta chain
    • Complex: Tubulin polyglutamylase TTLL6 in complex with unmodified human microtubule
      • Other: Tubulin polyglutamylase TTLL6

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Supramolecule #1: TTLL6 bound to unmodified human microtubule

SupramoleculeName: TTLL6 bound to unmodified human microtubule / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Microtubules were decorated with TTLL6 on electron microscopy grids
Molecular weightTheoretical: 100 KDa

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Supramolecule #2: alpha1B and betaI/betaIVb microtubule

SupramoleculeName: alpha1B and betaI/betaIVb microtubule / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human) / Organ: kidney / Location in cell: cytoplasm

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Supramolecule #3: Tubulin polyglutamylase TTLL6 in complex with unmodified human mi...

SupramoleculeName: Tubulin polyglutamylase TTLL6 in complex with unmodified human microtubule
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Mus musculus (house mouse) / Location in cell: cytoplasm

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: other / ID: 1 / Classification: other
Source (natural)Organism: Homo sapiens (human) / Organ: kidney / Tissue: kidney
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL D RIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE FSIYPAPQVS TA VVEPYNS ILTTHTTLEH SDCAFMVDNE AIYDICRRNL DIERPTYTNL NRLISQIVSS ITA SLRFDG ALNVDLTEFQ TNLVPYPRIH FPLATYAPVI SAEKAYHEQL SVAEITNACF EPAN QMVKC DPRHGKYMAC CLLYRGDVVP KDVNAAIATI KTKRSIQFVD WCPTGFKVGI NYQPP TVVP GGDLAKVQRA VCMLSNTTAI AEAWARLDHK FDLMYAKRAF VHWYVGEGME EGEFSE ARE DMAALEKDYE EVGVDSVEGE GEEEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: other / ID: 2 / Classification: other
Source (natural)Organism: Homo sapiens (human) / Organ: kidney / Tissue: kidney
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV V RKEAESCD CLQGFQLTHS LGGGTGSGMG TLLISKIREE YPDRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLDRISVY YNEATGGKYV PRAILVDLE PGTMDSVRSG PFGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV V RKEAESCD CLQGFQLTHS LGGGTGSGMG TLLISKIREE YPDRIMNTFS VVPSPKVSDT VV EPYNATL SVHQLVENTD ETYCIDNEAL YDICFRTLKL TTPTYGDLNH LVSATMSGVT TCL RFPGQL NADLRKLAVN MVPFPRLHFF MPGFAPLTSR GSQQYRALTV PELTQQVFDA KNMM AACDP RHGRYLTVAA VFRGRMSMKE VDEQMLNVQN KNSSYFVEWI PNNVKTAVCD IPPRG LKMA VTFIGNSTAI QELFKRISEQ FTAMFRRKAF LHWYTGEGMD EMEFTEAESN MNDLVS EYQ QYQDATAEEE EDFGEEAEEE A

UniProtKB: Tubulin beta chain

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Macromolecule #3: Tubulin polyglutamylase TTLL6

MacromoleculeName: Tubulin polyglutamylase TTLL6 / type: other / ID: 3 / Classification: other
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: GKKKRKKKRL VINLSNCRYD SVRRAAQQYG LREAGDNDDW TLYWTDYSVS LERVMEMKSY QKINHFPGMS EICRKDLLAR NMSRMLKLFP KDFHFFPRTW CLPADWGDLQ TYSRTRKNKT YICKPDSGCQ GRGIFITRSV KEIKPGEDMI CQLYISKPFI IDGFKFDLRV ...String:
GKKKRKKKRL VINLSNCRYD SVRRAAQQYG LREAGDNDDW TLYWTDYSVS LERVMEMKSY QKINHFPGMS EICRKDLLAR NMSRMLKLFP KDFHFFPRTW CLPADWGDLQ TYSRTRKNKT YICKPDSGCQ GRGIFITRSV KEIKPGEDMI CQLYISKPFI IDGFKFDLRV YVLVTSCDPL RVFVYNEGLA RFATTSYSHP NLDNLDEICM HLTNYSINKH SSNFVQDAFS GSKRKLSTFN SYMKTHGYDV EQIWRGIEDV IIKTLISAHP VIKHNYHTCF PSHTLNSACF EILGFDILLD RKLKPWLLEV NHSPSFSTDS KLDKEVKDSL LYDALVLINL GNCDKKKVLE EERQRGRFLQ QCPNREIRLE EVKGFQAMRL QKTEEYEKKN CGGFRLIYPG LNLEKYDKFF QDNSSLFQNT VASRARELYA RQLIQELRQK QEKKVFLKKA RKA

UniProtKB: Tubulin polyglutamylase TTLL6
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.1 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
40.0 mMC8H18N2O4SHEPES
100.0 mMKClPotassium chloride
20.0 mMMgCl2Magnesium chloride
4.0 mMDTTDithiothreitol
20.0 uMATPAdenosine triphosphateAdenosine triphosphate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 100
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 303 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was blotted with Whatman #5 blotting paper on both sides of the grid for 3 s with a blot offset of -1 using a Vitrobot Mark IV (Thermo Fisher Scientific) with a chamber set to 30C ...Details: The sample was blotted with Whatman #5 blotting paper on both sides of the grid for 3 s with a blot offset of -1 using a Vitrobot Mark IV (Thermo Fisher Scientific) with a chamber set to 30C at 100% humidity and subsequently plunged into liquid ethane..
DetailsTTLL6 decoration of microtubule was heterogenous

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-39 / Number real images: 2771 / Average exposure time: 9.75 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 59044
Details: Due to highly heterogeneous TTLL6 decoration of microtubules microtubules were manually selected. 59044 overlapping segments with a box size of 568 pixels were extracted every 82 A
Startup modelType of model: OTHER
Details: synthetic microtubule references were generated in UCSF Chimera
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 392289
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5n5n

residue_range: 1-437, source_name: PDB, initial_model_type: experimental model

6vzu

chain_id: A, residue_range: 57-461, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation
Output model

PDB-8u3z:
Model of TTLL6 bound to microtubule from composite map

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