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Yorodumi- EMDB-71753: Cryo-ET reconstruction of a regenerating axon after axotomy showi... -
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Basic information
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| Title | Cryo-ET reconstruction of a regenerating axon after axotomy showing polymerizing microtubules (primary mouse thalamus neuronal explant) | |||||||||
Map data | tomogram | |||||||||
Sample |
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Keywords | cytoskeleton / microtubules / neuroregeneration / axon / STRUCTURAL PROTEIN | |||||||||
| Biological species | ![]() | |||||||||
| Method | electron tomography / cryo EM | |||||||||
Authors | Bodakuntla S / Taira K / Yamada Y / Alvarez-Brecht P / Cada AK / Basnet N / Zhang R / Martinez-Sanchez A / Biertumpfel C / Mizuno N | |||||||||
| Funding support | United States, 1 items
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Citation | Journal: Nature / Year: 2025Title: 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. | |||||||||
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Structure visualization
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Downloads & links
-EMDB archive
| Map data | emd_71753.map.gz | 2.4 GB | EMDB map data format | |
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| Header (meta data) | emd-71753-v30.xml emd-71753.xml | 15.2 KB 15.2 KB | Display Display | EMDB header |
| Images | emd_71753.png | 195.7 KB | ||
| Filedesc metadata | emd-71753.cif.gz | 4.4 KB | ||
| Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-71753 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-71753 | HTTPS FTP |
-Related structure data
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Links
| EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Map
| File | Download / File: emd_71753.map.gz / Format: CCP4 / Size: 2.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||
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| Annotation | tomogram | ||||||||||||||||||||||||||||||||
| Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
| Voxel size | X=Y=Z: 10.68 Å | ||||||||||||||||||||||||||||||||
| Density |
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| Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
| Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : Thalamus primary tissue, neuron
| Entire | Name: Thalamus primary tissue, neuron |
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| Components |
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-Supramolecule #1: Thalamus primary tissue, neuron
| Supramolecule | Name: Thalamus primary tissue, neuron / type: cell / ID: 1 / Parent: 0 Details: Explant axon from thalamus primary tissue of mouse embryo |
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| Source (natural) | Organism: ![]() |
-Experimental details
-Structure determination
| Method | cryo EM |
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Processing | electron tomography |
| Aggregation state | cell |
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Sample preparation
| Buffer | pH: 7.2 / Details: Gibco Neurobasal Medium |
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| Grid | Model: Quantifoil R1/4 / Material: GOLD / Mesh: 200 / Support film - Material: SILICON DIOXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 3.8000000000000003 kPa / Details: coated with poly-L-lysine and laminin |
| Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV |
| Cryo protectant | No |
| Sectioning | Other: NO SECTIONING |
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Electron microscopy
| Microscope | TFS KRIOS |
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| Specialist optics | Energy filter - Name: GIF Bioquantum |
| Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 61 / Average electron dose: 125.0 e/Å2 |
| Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
| Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 33000 |
| Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
| Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
| Final reconstruction | Algorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 61 |
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| CTF correction | Software - Name: CTFPHASEFLIP / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Keywords
Authors
United States, 1 items
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FIELD EMISSION GUN
