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- PDB-9f3r: 13pf E254Q microtubule from recombinant human tubulin decorated w... -

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Basic information

Entry
Database: PDB / ID: 9f3r
Title13pf E254Q microtubule from recombinant human tubulin decorated with EB3
Components
  • Detyrosinated tubulin alpha-1B chain
  • Microtubule-associated protein RP/EB family member 3
  • Tubulin beta-3 chain
KeywordsSTRUCTURAL PROTEIN / Microtubule Tubulin GTP cp Cell cycle
Function / homology
Function and homology information


netrin receptor binding / mitotic spindle astral microtubule end / Post-chaperonin tubulin folding pathway / protein localization to microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end ...netrin receptor binding / mitotic spindle astral microtubule end / Post-chaperonin tubulin folding pathway / protein localization to microtubule / dorsal root ganglion development / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cilium Assembly / cytoskeleton-dependent intracellular transport / Carboxyterminal post-translational modifications of tubulin / microtubule plus-end / Intraflagellar transport / Sealing of the nuclear envelope (NE) by ESCRT-III / Formation of tubulin folding intermediates by CCT/TriC / microtubule plus-end binding / COPI-independent Golgi-to-ER retrograde traffic / Gap junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / Kinesins / Assembly and cell surface presentation of NMDA receptors / COPI-dependent Golgi-to-ER retrograde traffic / intercellular bridge / regulation of microtubule polymerization / microtubule organizing center / Recycling pathway of L1 / positive regulation of cyclin-dependent protein serine/threonine kinase activity / RHOH GTPase cycle / positive regulation of protein kinase activity / spindle midzone / regulation of microtubule polymerization or depolymerization / RHO GTPases activate IQGAPs / cytoplasmic microtubule / Hedgehog 'off' state / COPI-mediated anterograde transport / spindle assembly / microtubule-based process / Activation of AMPK downstream of NMDARs / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / MHC class II antigen presentation / Recruitment of NuMA to mitotic centrosomes / Resolution of Sister Chromatid Cohesion / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / cellular response to interleukin-4 / peptide binding / axon guidance / filopodium / cell periphery / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / PKR-mediated signaling / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / HCMV Early Events / Aggrephagy / Separation of Sister Chromatids / microtubule cytoskeleton / The role of GTSE1 in G2/M progression after G2 checkpoint / protein localization / mitotic cell cycle / lamellipodium / double-stranded RNA binding / growth cone / midbody / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / axon / cell division / GTPase activity / neuronal cell body / dendrite / ubiquitin protein ligase binding / protein kinase binding / GTP binding / positive regulation of DNA-templated transcription / perinuclear region of cytoplasm / structural molecule activity / extracellular exosome / identical protein binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Alpha tubulin ...EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) 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
GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1B chain / Tubulin beta-3 chain / Microtubule-associated protein RP/EB family member 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsEstevez-Gallego, J. / Blum, T.B. / Steinmetz, M.O. / Surrey, T.
Funding support Spain, Switzerland, European Union, 5items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesPID2019-108415GB-I00 Spain
Swiss National Science Foundation310030_192566 Switzerland
Generalitat de CatalunyaIU16-014045 (CRYO-TEM) Spain
European Research Council (ERC)951430European Union
Spanish Ministry of Science, Innovation, and UniversitiesFJC2020-043857-I Spain
CitationJournal: Nat Commun / Year: 2025
Title: Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap.
Authors: Juan Estévez-Gallego / Thorsten B Blum / Felix Ruhnow / María Gili / Silvia Speroni / Raquel García-Castellanos / Michel O Steinmetz / Thomas Surrey /
Abstract: A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis- ...A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis-deficient tubulin mutants have been used as GTP-cap mimics for structural and biochemical studies. However, these analogs and mutants generate microtubules with diverse biochemical properties and lattice structures, leaving it unclear what is the most faithful GTP mimic and hence the structure of the GTP cap. Here, we generate a hydrolysis-deficient human tubulin mutant, αE254Q, with the smallest possible modification. We show that αE254Q-microtubules are stable, but still exhibit mild mutation-induced growth abnormalities. However, mixing two GTP hydrolysis-deficient tubulin mutants, αE254Q and αE254N, at an optimized ratio eliminates growth and lattice abnormalities, indicating that these 'mosaic microtubules' are faithful GTP cap mimics. Their cryo-electron microscopy structure reveals that longitudinal lattice expansion, but not protofilament twist, is the primary structural feature distinguishing the GTP-tubulin containing cap from the GDP-tubulin containing microtubule shaft. However, alterations in protofilament twist may be transiently needed to allow lattice compaction and GTP hydrolysis. Together, our results provide insights into the structural origin of GTP cap stability, the pathway of GTP hydrolysis and hence microtubule dynamic instability.
History
DepositionApr 25, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
S: Microtubule-associated protein RP/EB family member 3
T: Microtubule-associated protein RP/EB family member 3
A: Detyrosinated tubulin alpha-1B chain
B: Tubulin beta-3 chain
G: Detyrosinated tubulin alpha-1B chain
N: Tubulin beta-3 chain
C: Detyrosinated tubulin alpha-1B chain
D: Tubulin beta-3 chain
I: Detyrosinated tubulin alpha-1B chain
P: Tubulin beta-3 chain
E: Detyrosinated tubulin alpha-1B chain
F: Tubulin beta-3 chain
K: Detyrosinated tubulin alpha-1B chain
R: Tubulin beta-3 chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)649,39538
Polymers642,82514
Non-polymers6,57024
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

#1: Protein Microtubule-associated protein RP/EB family member 3 / EB1 protein family member 3 / EBF3 / End-binding protein 3 / EB3 / RP3


Mass: 15386.819 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MAPRE3 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UPY8
#2: Protein
Detyrosinated tubulin alpha-1B chain


Mass: 50732.238 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBA1B / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P68363
#3: Protein
Tubulin beta-3 chain / Tubulin beta-4 chain / Tubulin beta-III


Mass: 51276.367 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TUBB3, TUBB4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q13509
#4: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 12 / 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.: 12 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: helical reconstruction

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Sample preparation

ComponentName: 13pf E254Q microtubule from recombinant human tubulin decorated with EB3
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 6.8
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 310 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 7.19 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2954

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Processing

EM software
IDNameVersionCategory
1RELION3.1.2particle selection
4RELION3.1.2CTF correction
7Coot0.9.8.92model fitting
9PHENIX1.21model refinement
12RELION3.1.2classification
13PHENIX1.213D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -27.6835 ° / Axial rise/subunit: 9.58171 Å / Axial symmetry: C13
Particle selectionNum. of particles selected: 790270
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33410 / Symmetry type: HELICAL
Atomic model buildingB value: 190 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7sj7

7sj7


Accession code: 7sj7 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 32.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005144256
ELECTRON MICROSCOPYf_angle_d0.739360082
ELECTRON MICROSCOPYf_chiral_restr0.0496552
ELECTRON MICROSCOPYf_plane_restr0.00617794
ELECTRON MICROSCOPYf_dihedral_angle_d11.59336068

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