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- EMDB-42884: Microtubule-TTLL6 map -

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Basic information

Entry
Database: EMDB / ID: EMD-42884
TitleMicrotubule-TTLL6 map
Map dataMap of alpha1B and betaI and betaIVb microtubule, locally sharpened
Sample
  • Complex: TTLL6 bound to unmodified human microtubules
    • Complex: Tubulin alpha-1B, betaI and betaIVb
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta-I/IVb chain
    • Complex: Tubulin polyglutamylase TTLL6
      • Protein or peptide: Tubulin polyglutamylase TTLL6
Keywordstubulin post-translational modifications / microtubules / TTLL / LIGASE
Function / homology
Function and homology information


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 / cytoskeleton-dependent intracellular transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC ...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 / cytoskeleton-dependent intracellular transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / natural killer cell mediated cytotoxicity / Kinesins / Assembly and cell surface presentation of NMDA receptors / 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 / RHOH GTPase cycle / spindle assembly / RHO GTPases activate IQGAPs / cellular response to interleukin-4 / MHC class I protein binding / Hedgehog 'off' state / microtubule-based process / 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 / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / MHC class II antigen presentation / AURKA Activation by TPX2 / 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 / structural constituent of cytoskeleton / cytoplasmic ribonucleoprotein granule / mitotic spindle / microtubule cytoskeleton organization / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / microtubule cytoskeleton / azurophil granule lumen / double-stranded RNA binding / Regulation of PLK1 Activity at G2/M Transition / 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 / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
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-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 beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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
DepositionNov 20, 2023-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42884.png

Downloads & links

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Map

FileDownload / File: emd_42884.map.gz / Format: CCP4 / Size: 699 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of alpha1B and betaI and betaIVb microtubule, locally sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 568 pix.
= 653.2 Å		1.15 Å/pix.
x 568 pix.
= 653.2 Å		1.15 Å/pix.
x 568 pix.
= 653.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.005091535 - 0.13096036
Average (Standard dev.)0.00005778015 (±0.0014560403)
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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Mask #1

Fileemd_42884_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw map of alpha1B and betaI and betaIVb microtubule

Fileemd_42884_additional_1.map
AnnotationRaw map of alpha1B and betaI and betaIVb microtubule
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map of alpha1B and betaI and betaIVb microtubule,...

Fileemd_42884_additional_2.map
AnnotationMap of alpha1B and betaI and betaIVb microtubule, post-processed with DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second half-map of alpha1B and betaI and betaIVb microtubule

Fileemd_42884_half_map_1.map
AnnotationSecond half-map of alpha1B and betaI and betaIVb microtubule
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First half-map of alpha1B and betaI and betaIVb microtubule

Fileemd_42884_half_map_2.map
AnnotationFirst half-map of alpha1B and betaI and betaIVb microtubule
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 microtubules

EntireName: TTLL6 bound to unmodified human microtubules
Components
  • Complex: TTLL6 bound to unmodified human microtubules
    • Complex: Tubulin alpha-1B, betaI and betaIVb
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta-I/IVb chain
    • Complex: Tubulin polyglutamylase TTLL6
      • Protein or peptide: Tubulin polyglutamylase TTLL6

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Supramolecule #1: TTLL6 bound to unmodified human microtubules

SupramoleculeName: TTLL6 bound to unmodified human microtubules / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Microtubules were decorated with TTLL6 on electron microscopy grids
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: Tubulin alpha-1B, betaI and betaIVb

SupramoleculeName: Tubulin alpha-1B, betaI and betaIVb / 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

SupramoleculeName: Tubulin polyglutamylase TTLL6 / 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: protein_or_peptide / ID: 1 / Details: a-tubulin from tsA201 cell line / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: kidney
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta-I/IVb chain

MacromoleculeName: Tubulin beta-I/IVb chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: 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: protein_or_peptide / ID: 3 / Enantiomer: LEVO
EC number: Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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

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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 mMDithiothreitol
20.0 uMAdenosine triphosphate
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: 3.6 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
source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation

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