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- PDB-8t42: Model of TTLL6 MTBH1-2 bound to microtubule -

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Basic information

Entry
Database: PDB / ID: 8t42
TitleModel of TTLL6 MTBH1-2 bound to microtubule
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
  • Tubulin polyglutamylase TTLL6
KeywordsLIGASE / tubulin post-translational modifications / microtubules / TTLL
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 / cytoskeleton-dependent intracellular transport / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / COPI-independent Golgi-to-ER retrograde traffic / natural killer cell mediated cytotoxicity / microtubule bundle formation / Kinesins / Assembly and cell surface presentation of NMDA receptors / GTPase activating protein binding / COPI-dependent Golgi-to-ER retrograde traffic / regulation of synapse organization / intercellular bridge / nuclear envelope lumen / Recycling pathway of L1 / RHOH GTPase cycle / RHO GTPases activate IQGAPs / spindle assembly / MHC class I protein binding / Hedgehog 'off' state / cytoplasmic microtubule / 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 / cellular response to interleukin-4 / 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 / 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 / Regulation of PLK1 Activity at G2/M Transition / double-stranded RNA binding / mitotic cell cycle / cell body / microtubule / Potential therapeutics for SARS / cytoskeleton / membrane raft / cell division / protein domain specific binding / 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
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin polyglutamylase TTLL6 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMahalingan, K.K. / Grotjahn, D. / Li, Y. / Lander, G.C. / Zehr, E.A. / Roll-Mecak, A.
Funding support United States, 1items
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 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
E: Tubulin beta chain
F: Tubulin alpha-1B chain
N: Tubulin polyglutamylase TTLL6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)261,17613
Polymers258,9905
Non-polymers2,1868
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 5 molecules AFBEN

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50204.445 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: a-tubulin from tsA201 cell line / Source: (natural) Homo sapiens (human) / Cell: Epithelial-like / Cell line: tsA201 / Organ: kidney / Tissue: kidney / References: UniProt: P68363
#2: Protein Tubulin beta chain / Tubulin beta-5 chain


Mass: 49717.629 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell: Epithelial-like / Cell line: tsA201 / Organ: kidney / Tissue: kidney / References: UniProt: P07437
#3: Protein Tubulin polyglutamylase TTLL6 / Protein polyglutamylase TTLL6 / Tubulin--tyrosine ligase-like protein 6


Mass: 59145.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ttll6 / Plasmid: P7XNH3 / Production host: Escherichia coli (E. coli) / Strain (production host): Arctic Express DE3
References: UniProt: A4Q9E8, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)

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Non-polymers , 3 types, 8 molecules

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


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1TTLL6 bound to unmodified human microtubulesCOMPLEXMicrotubules were decorated with TTLL6 on electron microscopy grids#1-#30MULTIPLE SOURCES
2Tubulin alpha-1B chain, Tubulin betaI chainCOMPLEX#1-#21NATURAL
3Tubulin polyglutamylase TTLL6COMPLEX#31RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrgan
12Homo sapiens (human)9606cytoplasmkidney
23Mus musculus (house mouse)10090cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Arctic Express DE3 / Plasmid: P7XNH3
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
140 mMHEPESC8H18N2O4S1
2100 mMPotassium chlorideKCl1
320 mMMagnesium chlorideMgCl21
44 mMDithiothreitolDTT1
520 uMAdenosine triphosphateATPAdenosine triphosphate1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: TTLL6 decoration of microtubule was heterogenous
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 303 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Image recordingAverage exposure time: 9.75 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2771
Image scansMovie frames/image: 39 / Used frames/image: 1-39

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 392289 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain residue rangeInitial refinement model-IDPdb chain residue rangeSource nameTypeChain-ID
15N5N

5n5n

5N5N1-43711-437PDBexperimental model
26VZU

6vzu

A6VZU57-461257-461PDBexperimental modelA
3AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00714175
ELECTRON MICROSCOPYf_angle_d1.09719252
ELECTRON MICROSCOPYf_dihedral_angle_d9.2631938
ELECTRON MICROSCOPYf_chiral_restr0.0532108
ELECTRON MICROSCOPYf_plane_restr0.0092503

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