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- EMDB-41022: Map of Tubulin Tyrosine Ligase Like 6 -

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Basic information

Entry
Database: EMDB / ID: EMD-41022
TitleMap of Tubulin Tyrosine Ligase Like 6
Map dataMap of TTLL6 bound to unmodified human microtubules, obtained by focused classification and refinement
Sample
  • Complex: TTLL6 bound to unmodified human microtubules
    • Protein or peptide: Tubulin Tyrosine Ligase Like 6
KeywordsTubulin polyglutamylase / tubulin 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) / microtubule bundle formation ...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) / microtubule bundle formation / tubulin binding / ciliary basal body / cilium / microtubule cytoskeleton organization / microtubule / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin-tyrosine ligase/Tubulin polyglutamylase / Tubulin-tyrosine ligase family / TTL domain profile.
Similarity search - Domain/homology
Tubulin polyglutamylase TTLL6
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 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 8, 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_41022.png

Downloads & links

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Map

FileDownload / File: emd_41022.map.gz / Format: CCP4 / Size: 699 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of TTLL6 bound to unmodified human microtubules, obtained by focused classification and refinement
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.02678331 - 0.04489845
Average (Standard dev.)0.00000048974226 (±0.0001945757)
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_41022_msk_1.map
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...

Fileemd_41022_half_map_1.map
AnnotationFirst half-map of TTLL6 bound to unmodified human microtubules, obtained by focused classification and refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_41022_half_map_2.map
AnnotationSecond half-map of TTLL6 bound to unmodified human microtubules, obtained by focused classification and refinement
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
    • Protein or peptide: Tubulin Tyrosine Ligase Like 6

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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
Source (natural)Organism: Mus musculus (house mouse) / Location in cell: cytoplasm
Molecular weightTheoretical: 20 KDa

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Macromolecule #1: Tubulin Tyrosine Ligase Like 6

MacromoleculeName: Tubulin Tyrosine Ligase Like 6 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ec: 6.3.2.61
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli 0.1304 (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

UniProtKB: Tubulin polyglutamylase TTLL6

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
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: Map of TTLL6 bound to unmodified human microtubules
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final 3D classificationNumber classes: 2 / Avg.num./class: 196000 / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8)
Details: Map obtained through focused classification with subtraction of microtubule signal, followed by 3D refinement.
Number images used: 151716
FSC plot (resolution estimation)

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