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- EMDB-15438: In situ cryo-electron tomogram of an intact differentiated P19 ne... -

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Entry
Database: EMDB / ID: EMD-15438
TitleIn situ cryo-electron tomogram of an intact differentiated P19 neuronal process
Map dataIn situ cryo-electron tomogram of an intact differentiated P19 neuronal process
Sample
  • Cell: In situ cryo-electron tomogram of an intact differentiated P19 neuronal process
KeywordsMicrotubules / Scaffold / protein transport
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsChakraborty S / Martinez-Sanchez A / Baumeister W / Mahamid J
Funding support Germany, 1 items
OrganizationGrant numberCountry
Other government Germany
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2025
Title: Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules.
Authors: Saikat Chakraborty / Antonio Martinez-Sanchez / Florian Beck / Mauricio Toro-Nahuelpan / In-Young Hwang / Kyung-Min Noh / Wolfgang Baumeister / Julia Mahamid /
Abstract: The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. ...The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) organization and structures of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons, and pluripotent and differentiated P19 cells. We obtain in situ density maps of several lumenal particles from the respective cells and detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggest that a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggests a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.
#1: Journal: Biorxiv / Year: 2024
Title: Cryo-electron tomography suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules
Authors: Chakraborty S / Martinez-Sanchez A / Beck F / Toro-Nahuelpan M / Hwang IY / Noh KM / Baumeister W / Mahamid J
History
DepositionJul 21, 2022-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15438.png

Downloads & links

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Map

FileDownload / File: emd_15438.map.gz / Format: CCP4 / Size: 205.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIn situ cryo-electron tomogram of an intact differentiated P19 neuronal process
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.92 Å/pix.
x 250 pix.
= 4480. Å		17.92 Å/pix.
x 464 pix.
= 8314.88 Å		17.92 Å/pix.
x 464 pix.
= 8314.88 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 17.92 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-39.731876 - 41.176215999999997
Average (Standard dev.)1.2204754 (±5.0371284)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-125
Dimensions464464250
Spacing464464250
CellA: 8314.88 Å / B: 8314.88 Å / C: 4480.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : In situ cryo-electron tomogram of an intact differentiated P19 ne...

EntireName: In situ cryo-electron tomogram of an intact differentiated P19 neuronal process
Components
  • Cell: In situ cryo-electron tomogram of an intact differentiated P19 neuronal process

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Supramolecule #1: In situ cryo-electron tomogram of an intact differentiated P19 ne...

SupramoleculeName: In situ cryo-electron tomogram of an intact differentiated P19 neuronal process
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.2
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV
Details: blotted from backside with blot force 10 and blot time 10 seconds.
DetailsFrozen hydrated differentiated P19 neurons.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsDose symmetric acquisition scheme
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus min: 6.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 64000
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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9.0) / Number images used: 40

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