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- EMDB-71840: Cryo-ET subtomogram averaging of a stress fiber from a regenerati... -

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Basic information

Entry
Database: EMDB / ID: EMD-71840
TitleCryo-ET subtomogram averaging of a stress fiber from a regenerating axon
Map datapostprocessed map from subtomogram averaging
Sample
  • Cell: Thalamus primary tissue, neuron
Keywordscytoskeleton / F-actin / neuroregeneration / axon / STRUCTURAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 27.7 Å
AuthorsBodakuntla 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
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 28, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 3, 2025-
Current statusDec 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71840.png

Downloads & links

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Map

FileDownload / File: emd_71840.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed map from subtomogram averaging
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.68 Å/pix.
x 192 pix.
= 513.6 Å		2.68 Å/pix.
x 192 pix.
= 513.6 Å		2.68 Å/pix.
x 192 pix.
= 513.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.675 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.015425046 - 0.110328004
Average (Standard dev.)0.00077592133 (±0.006654625)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 513.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_71840_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from Refine3D

Fileemd_71840_half_map_1.map
Annotationhalf map from Refine3D
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map from Refine3D

Fileemd_71840_half_map_2.map
Annotationhalf map from Refine3D
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thalamus primary tissue, neuron

EntireName: Thalamus primary tissue, neuron
Components
  • Cell: Thalamus primary tissue, neuron

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Supramolecule #1: Thalamus primary tissue, neuron

SupramoleculeName: Thalamus primary tissue, neuron / type: cell / ID: 1 / Parent: 0
Details: Explant axon from thalamus primary tissue of mouse embryo
Source (natural)Organism: Mus musculus (house mouse) / Organ: Brain / Tissue: Thalamus

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.2 / Details: Gibco Neurobasal Medium
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 61 / Average electron dose: 125.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 33000
Sample stageSpecimen 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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 27.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Number subtomograms used: 1
ExtractionNumber tomograms: 1 / Number images used: 1555 / Reference model: cylinder / Software - Name: IMOD
CTF correctionSoftware - Name: RELION (ver. 5) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Final 3D classificationNumber classes: 1 / Avg.num./class: 1555 / Software - Name: RELION (ver. 5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5)
FSC plot (resolution estimation)

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