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- PDB-9pnd: In situ microtubule of EpoB-induced regenerating axons -

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Basic information

Entry
Database: PDB / ID: 9pnd
TitleIn situ microtubule of EpoB-induced regenerating axons
Components
  • Detyrosinated tubulin alpha-1A chain
  • Tubulin beta-3 chain
KeywordsSTRUCTURAL PROTEIN / cytoskeleton / microtubules / neuroregeneration / axon
Function / homology
Function and homology information


Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cargo trafficking to the periciliary membrane / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / netrin-activated signaling pathway / COPI-independent Golgi-to-ER retrograde traffic / netrin receptor binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-mediated anterograde transport ...Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Cargo trafficking to the periciliary membrane / Sealing of the nuclear envelope (NE) by ESCRT-III / Intraflagellar transport / Carboxyterminal post-translational modifications of tubulin / netrin-activated signaling pathway / COPI-independent Golgi-to-ER retrograde traffic / netrin receptor binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-mediated anterograde transport / Kinesins / PKR-mediated signaling / Aggrephagy / RHO GTPases activate IQGAPs / COPI-dependent Golgi-to-ER retrograde traffic / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / Recycling pathway of L1 / The role of GTSE1 in G2/M progression after G2 checkpoint / dorsal root ganglion development / axonemal microtubule / Hedgehog 'off' state / RHO GTPases Activate Formins / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / organelle transport along microtubule / Anchoring of the basal body to the plasma membrane / Separation of Sister Chromatids / Recruitment of NuMA to mitotic centrosomes / AURKA Activation by TPX2 / forebrain morphogenesis / glial cell differentiation / cerebellar cortex morphogenesis / dentate gyrus development / Regulation of PLK1 Activity at G2/M Transition / neuron projection arborization / MHC class II antigen presentation / smoothened signaling pathway / motor behavior / pyramidal neuron differentiation / response to L-glutamate / centrosome cycle / adult behavior / 'de novo' protein folding / intercellular bridge / startle response / flagellated sperm motility / regulation of synapse organization / microtubule polymerization / locomotory exploration behavior / response to tumor necrosis factor / homeostasis of number of cells within a tissue / response to mechanical stimulus / sperm flagellum / adult locomotory behavior / neurogenesis / cytoplasmic microtubule / condensed chromosome / peptide binding / ciliary tip / cellular response to calcium ion / axon guidance / hippocampus development / locomotory behavior / cell periphery / filopodium / sperm principal piece / sperm end piece / neuromuscular junction / myelin sheath / neuron migration / cerebral cortex development / visual learning / intracellular protein transport / memory / synapse organization / microtubule cytoskeleton organization / recycling endosome / neuron apoptotic process / mitotic spindle / gene expression / structural constituent of cytoskeleton / neuron differentiation / cytoplasmic ribonucleoprotein granule / microtubule cytoskeleton / mitotic cell cycle / lamellipodium / growth cone / protein-folding chaperone binding / cilium / microtubule binding / microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / protein stabilization / membrane raft / protein heterodimerization activity / protein domain specific binding / axon
Similarity search - Function
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 ...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
Chem-EPB / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Tubulin alpha-1A chain / Tubulin beta-3 chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsBodakuntla, S. / Taira, K. / Yamada, Y. / Alvarez-Brecht, P. / Cada, A.K. / Basnet, N. / Zhang, R. / Martinez-Sanchez, A. / Biertumpfel, C. / Mizuno, N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1ZIAHL006264 United States
CitationJournal: Nature / Year: 2025
Title: In situ structural mechanism of epothilone-B-induced CNS axon regeneration.
Authors: Satish Bodakuntla / Kenichiro Taira / Yurika Yamada / Pelayo Alvarez-Brecht / A King Cada / Nirakar Basnet / Rui Zhang / Antonio Martinez-Sanchez / Christian Biertümpfel / Naoko Mizuno /
Abstract: Axons in the adult central nervous system (CNS) do not regenerate following injury, in contrast to neurons in the peripheral nervous system and neuronal growth during embryonic development. The ...Axons in the adult central nervous system (CNS) do not regenerate following injury, in contrast to neurons in the peripheral nervous system and neuronal growth during embryonic development. The molecular mechanisms that prevent regeneration of neurons in the CNS remain largely unknown. Here, to address the intracellular response to injury, we developed an in situ cryo-electron tomography and cryo-electron microscopy platform to mimic axonal damage and present the structural mechanism underlying thalamic axon regeneration induced by the drug epothilone B. We observed that stabilized microtubules extend beyond the injury site, generating membrane tension and driving membrane expansion. Cryo-electron microscopy reveals the in situ structure of microtubules at 3.19 Å resolution, which engage epothilone B within the microtubule lattice at the regenerating front. During repair, tubulin clusters are delivered and incorporated into polymerizing microtubules at the regenerating site. These microtubule shoots serve as scaffolds for various types of vesicles and endoplasmic reticulum, facilitating the supply of materials necessary for axon repair until membrane tension normalizes. We demonstrate the unexpected ability of neuronal cells to adjust to strain induced by epothilone B, which creates homeostatic imbalances and activates axons to regeneration mode.
History
DepositionJul 20, 2025Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Tubulin beta-3 chain
D: Tubulin beta-3 chain
A: Detyrosinated tubulin alpha-1A chain
C: Detyrosinated tubulin alpha-1A chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,35714
Polymers201,3124
Non-polymers3,04510
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, negative-staining EM, assay for oligomerization, absorbance at 400 nm
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules BDAC

#1: Protein Tubulin beta-3 chain


Mass: 50467.492 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: TUBB3 component of microtubules / Source: (natural) Mus musculus (house mouse) / Organ: Brain / Plasmid details: Axon / Tissue: Thalamus / References: UniProt: Q9ERD7
#2: Protein Detyrosinated tubulin alpha-1A chain


Mass: 50188.441 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Details: TUBA1A component of microtubules / Source: (natural) Mus musculus (house mouse) / Organ: Brain / Plasmid details: Axon / Tissue: Thalamus / References: UniProt: P68369

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Non-polymers , 5 types, 14 molecules

#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical ChemComp-EPB / 7,11-DIHYDROXY-8,8,10,12,16-PENTAMETHYL-3-[1-METHYL-2-(2-METHYL-THIAZOL-4-YL)VINYL]-4,17-DIOXABICYCLO[14.1.0]HEPTADECANE-5,9-DIONE / EPOTHILONE B


Mass: 507.683 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H41NO6S / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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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: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Microtubule, axonal / Type: ORGANELLE OR CELLULAR COMPONENT
Details: from Epothilone B-induced regenerating explant axons from thalamus primary mouse embryo tissue after axotomy
Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse) / Cellular location: Axon / Organ: Brain / Tissue: Thalamus
Buffer solutionpH: 7.2 / Details: Gibco Neurobasal Media
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: induced by axotomy in the presence of 1 nM Epothilone B
Specimen supportDetails: coated with poly-L-lysine and laminin / Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1/4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 5 / Num. of real images: 4034 / Details: curated image number
EM imaging opticsEnergyfilter name: GIF Bioquantum
Image scansWidth: 11520 / Height: 8184

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5particle selection
2SerialEMimage acquisition
4cryoSPARC4.5CTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11cryoSPARC4.5initial Euler assignment
12cryoSPARC4.5final Euler assignment
13cryoSPARC4.5classification
14cryoSPARC4.53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -27.64 ° / Axial rise/subunit: 9.695 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 399396
Details: picked in filament tracer with overlapping boxes with a step size of 82.5 Angstrom
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118551 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6dpu

6dpu


Accession code: 6dpu / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.19 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00114088
ELECTRON MICROSCOPYf_angle_d0.44219142
ELECTRON MICROSCOPYf_dihedral_angle_d10.6622044
ELECTRON MICROSCOPYf_chiral_restr0.0392092
ELECTRON MICROSCOPYf_plane_restr0.0032486

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