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- PDB-8yal: ATAT-2 bound K40Q MEC-12/MEC-7 microtubule -

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Basic information

Entry
Database: PDB / ID: 8yal
TitleATAT-2 bound K40Q MEC-12/MEC-7 microtubule
Components
  • Alpha-tubulin N-acetyltransferase 2
  • Tubulin alpha-3 chain
  • Tubulin beta-1 chain
KeywordsSTRUCTURAL PROTEIN / Microtubule / luminal enzyme / PTM
Function / homology
Function and homology information


positive regulation of detection of mechanical stimulus involved in sensory perception of touch / positive regulation of mechanosensory behavior / COPI-mediated anterograde transport / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / Intraflagellar transport / alpha-tubulin N-acetyltransferase / tubulin N-acetyltransferase activity / detection of mechanical stimulus involved in sensory perception of touch ...positive regulation of detection of mechanical stimulus involved in sensory perception of touch / positive regulation of mechanosensory behavior / COPI-mediated anterograde transport / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / Intraflagellar transport / alpha-tubulin N-acetyltransferase / tubulin N-acetyltransferase activity / detection of mechanical stimulus involved in sensory perception of touch / thigmotaxis / detection of mechanical stimulus involved in sensory perception / mechanosensory behavior / lysine N-acetyltransferase activity, acting on acetyl phosphate as donor / microtubule-based process / neuron development / regulation of microtubule cytoskeleton organization / cytoplasmic microtubule organization / response to mechanical stimulus / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / neuron projection / axon / GTPase activity / neuronal cell body / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Gcn5-related N-acetyltransferase (GNAT) domain, ATAT-type / Alpha-tubulin N-acetyltransferase / GNAT acetyltransferase, Mec-17 / Alpha-tubulin Gcn5-related N-acetyltransferase (GNAT) domain profile. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Gcn5-related N-acetyltransferase (GNAT) domain, ATAT-type / Alpha-tubulin N-acetyltransferase / GNAT acetyltransferase, Mec-17 / Alpha-tubulin Gcn5-related N-acetyltransferase (GNAT) domain profile. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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
ACETYL COENZYME *A / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / Tubulin beta-1 chain / Tubulin alpha-3 chain / Alpha-tubulin N-acetyltransferase 2
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLam, W.H. / Yu, D. / Zhai, Y. / Ti, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Tubulin acetyltransferases access and modify the microtubule luminal K40 residue through anchors in taxane-binding pockets.
Authors: Jingyi Luo / Wai Hei Lam / Daqi Yu / Victor C Chao / Marc Nicholas Zopfi / Chen Jing Khoo / Chang Zhao / Shan Yan / Zheng Liu / Xiang David Li / Chaogu Zheng / Yuanliang Zhai / Shih-Chieh Ti /
Abstract: Acetylation at α-tubulin K40 is the sole post-translational modification preferred to occur inside the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferases access the ...Acetylation at α-tubulin K40 is the sole post-translational modification preferred to occur inside the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferases access the luminal K40 in micrometer-long microtubules remains unknown. Here, we use cryo-electron microscopy and single-molecule reconstitution assays to reveal the enzymatic mechanism for tubulin acetyltransferases to modify K40 in the lumen. One tubulin acetyltransferase spans across the luminal lattice, with the catalytic core docking onto two α-tubulins and the enzyme's C-terminal domain occupying the taxane-binding pockets of two β-tubulins. The luminal accessibility and enzyme processivity of tubulin acetyltransferases are inhibited by paclitaxel, a microtubule-stabilizing chemotherapeutic agent. Characterizations using recombinant tubulins mimicking preacetylated and postacetylated K40 show the crosstalk between microtubule acetylation states and the cofactor acetyl-CoA in enzyme turnover. Our findings provide crucial insights into the conserved multivalent interactions involving α- and β-tubulins to acetylate the confined microtubule lumen.
History
DepositionFeb 9, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Tubulin alpha-3 chain
C: Alpha-tubulin N-acetyltransferase 2
D: Tubulin beta-1 chain
E: Tubulin alpha-3 chain
F: Tubulin beta-1 chain
I: Alpha-tubulin N-acetyltransferase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)262,71111
Polymers259,8126
Non-polymers2,8985
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 3 types, 6 molecules BECIDF

#1: Protein Tubulin alpha-3 chain / Mechanosensory abnormality protein 12 / MEC-12


Mass: 50165.367 Da / Num. of mol.: 2 / Mutation: K40Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mec-12, tba-3, C44B11.3 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P91910, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#2: Protein Alpha-tubulin N-acetyltransferase 2 / Alpha-TAT 2 / ATAT 2 / Mec-17-like protein


Mass: 30434.654 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: atat-2, W06B11.1 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q23192, alpha-tubulin N-acetyltransferase
#3: Protein Tubulin beta-1 chain / Beta-1-tubulin


Mass: 49306.176 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: MEC-7 / Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: mec-7, ZK154.3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P12456

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Non-polymers , 3 types, 5 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-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-ACO / ACETYL COENZYME *A


Mass: 809.571 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H38N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


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

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: K40Q MEC-12/MEC-7 microtubule complexed with alpha TAT2
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 6.8
Buffer component
IDConc.NameBuffer-ID
180 mMPotassium PIPES1
21 mMMagnesium chloride1
31 mMEGTA1
41 mMTCEP1
50.05 %IGEPAL1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 47170 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1100 nm / Calibrated defocus min: 1100 nm / Calibrated defocus max: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.1.2particle selection
2EPU2.12image acquisition
4cryoSPARC4.1.2CTF correction
7UCSF Chimera1.16model fitting
9PHENIX1.19-4092model refinement
10cryoSPARC4.1.2initial Euler assignment
11cryoSPARC4.1.2final Euler assignment
12cryoSPARC4.1.2classification
13cryoSPARC4.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 389554
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 370802 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315869
ELECTRON MICROSCOPYf_angle_d0.47921571
ELECTRON MICROSCOPYf_dihedral_angle_d4.8272159
ELECTRON MICROSCOPYf_chiral_restr0.0432358
ELECTRON MICROSCOPYf_plane_restr0.0042803

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