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- PDB-6i2i: Refined 13pf Hela Cell Tubulin microtubule (EML4-NTD decorated) -

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Basic information

Entry
Database: PDB / ID: 6i2i
TitleRefined 13pf Hela Cell Tubulin microtubule (EML4-NTD decorated)
Components
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsSTRUCTURAL PROTEIN / Microtubule / Tubulin / Hela / EML
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 / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / 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 / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Gap junction assembly / Formation of tubulin folding intermediates by CCT/TriC / natural killer cell mediated cytotoxicity / COPI-independent Golgi-to-ER retrograde traffic / 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 / spindle assembly / RHO GTPases activate IQGAPs / 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 / AURKA Activation by TPX2 / RHO GTPases Activate Formins / Translocation of SLC2A4 (GLUT4) to the plasma membrane / 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 / 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 / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal ...Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / 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 / Helix Hairpins / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsAtherton, J.M. / Moores, C.A.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust204801/Z/16/Z United Kingdom
16-0119 United Kingdom
Medical Research Council (United Kingdom)MR/R000352/1 United Kingdom
CitationJournal: Sci Signal / Year: 2019
Title: Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.
Authors: Rozita Adib / Jessica M Montgomery / Joseph Atherton / Laura O'Regan / Mark W Richards / Kees R Straatman / Daniel Roth / Anne Straube / Richard Bayliss / Carolyn A Moores / Andrew M Fry /
Abstract: EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the ...EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser and Ser in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.
History
DepositionNov 1, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,8697
Polymers99,9222
Non-polymers1,9475
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, Analysis by EM and microtubule co-sedimentation assay
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6100 Å2
ΔGint-50 kcal/mol
Surface area32930 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 50204.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela Cells / Plasmid details: Cytoskeleton Inc. / References: UniProt: P68363
#2: Protein Tubulin beta chain / Tubulin beta-5 chain


Mass: 49717.629 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Beta1-tubulin / Source: (natural) Homo sapiens (human) / Cell line: Hela Cells / Plasmid details: Cytoskeleton Inc. / References: UniProt: P07437

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Non-polymers , 4 types, 5 molecules

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


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-G2P / PHOSPHOMETHYLPHOSPHONIC ACID GUANYLATE ESTER


Mass: 521.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O13P3 / Comment: GMP-CPP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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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

ComponentName: Alpha and beta-tubulin from Hela Cell (modelled) decorated with EML4-NTD (not modelled)
Type: COMPLEX
Details: EML4-NTD density at low resolution due to flexibility, thus was not modelled
Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 110 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cell: Hela
Buffer solutionpH: 6.8
Details: 25mM PIPES, 1.5mM MgCl2, 1mM EGTA, 1mM DTT, 30mM NaCl,1mM GMPCPP
Buffer componentName: BRB25
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Microtubules formed from Hela cell tubulin
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Details: Dose-weighted sums used in reconstruction

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
7Cootmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19542 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00841940
ELECTRON MICROSCOPYf_angle_d1.04857072
ELECTRON MICROSCOPYf_dihedral_angle_d7.51325122
ELECTRON MICROSCOPYf_chiral_restr0.0616258
ELECTRON MICROSCOPYf_plane_restr0.0097686

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