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- EMDB-15444: Subtomogram average of floating microtubule inner protein 4 -

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Basic information

Entry
Database: EMDB / ID: EMD-15444
TitleSubtomogram average of floating microtubule inner protein 4
Map dataFloating microtubule inner protein 4
Sample
  • Cell: Floating microtubule inner protein 4 (fMIP4)
KeywordsMicrotubule-associated protein / STRUCTURAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 27.0 Å
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) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15444.png

Downloads & links

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Map

FileDownload / File: emd_15444.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFloating microtubule inner protein 4
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 128 pix.
= 437.76 Å		3.42 Å/pix.
x 128 pix.
= 437.76 Å		3.42 Å/pix.
x 128 pix.
= 437.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.46
Minimum - Maximum-4.743791 - 9.154636999999999
Average (Standard dev.)0.0050512003 (±0.27507618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 437.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Floating microtubule inner protein 4

Fileemd_15444_half_map_1.map
AnnotationFloating microtubule inner protein 4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Floating microtubule inner protein 4

Fileemd_15444_half_map_2.map
AnnotationFloating microtubule inner protein 4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Floating microtubule inner protein 4 (fMIP4)

EntireName: Floating microtubule inner protein 4 (fMIP4)
Components
  • Cell: Floating microtubule inner protein 4 (fMIP4)

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Supramolecule #1: Floating microtubule inner protein 4 (fMIP4)

SupramoleculeName: Floating microtubule inner protein 4 (fMIP4) / type: cell / ID: 1 / Parent: 0
Details: Subtomogram average was generated from in situ cryo-electron tomograms of pluripotent P19 cells
Source (natural)Organism: Mus musculus (house mouse)

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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
GridModel: Quantifoil R2/1 / Material: GOLD / 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: blot force 10, blot time 10 seconds from backside..
DetailsThese particles were found inside microtubule lumens in situ

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 77.0 K
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.4 e/Å2 / Details: bidirectional tilting scheme
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 42000
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: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number subtomograms used: 3060
ExtractionNumber tomograms: 46 / Number images used: 44944 / Reference model: De novo template free / Software - Name: IMOD (ver. 4.9.0)
Final 3D classificationSoftware - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
FSC plot (resolution estimation)

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