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- EMDB-13599: Tomogram of mouse dorsal root ganglion axon (dataset 2, TS_41). -

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Basic information

Entry
Database: EMDB / ID: EMD-13599
TitleTomogram of mouse dorsal root ganglion axon (dataset 2, TS_41).
Map dataTomogram of mouse dorsal root ganglion axon. The volume was binned by 4 and deconvolved for visualization.
Sample
  • Cell: Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2 (TS_41).
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsFoster HE / Ventura Santos C / Carter AP
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MRC_UP_A025_1011 United Kingdom
Wellcome TrustWT210711 United Kingdom
Citation
Journal: J Cell Biol / Year: 2022
Title: A cryo-ET survey of microtubules and intracellular compartments in mammalian axons.
Authors: Helen E Foster / Camilla Ventura Santos / Andrew P Carter /
Abstract: The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the ...The neuronal axon is packed with cytoskeletal filaments, membranes, and organelles, many of which move between the cell body and axon tip. Here, we used cryo-electron tomography to survey the internal components of mammalian sensory axons. We determined the polarity of the axonal microtubules (MTs) by combining subtomogram classification and visual inspection, finding MT plus and minus ends are structurally similar. Subtomogram averaging of globular densities in the MT lumen suggests they have a defined structure, which is surprising given they likely contain the disordered protein MAP6. We found the endoplasmic reticulum in axons is tethered to MTs through multiple short linkers. We surveyed membrane-bound cargos and describe unexpected internal features such as granules and broken membranes. In addition, we detected proteinaceous compartments, including numerous virus-like capsid particles. Our observations outline novel features of axonal cargos and MTs, providing a platform for identification of their constituents.
#1: Journal: Biorxiv / Year: 2021
Title: A cryo-ET survey of intracellular compartments within mammalian axons
Authors: Foster HE / Carter AP
History
DepositionSep 19, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_13599.png

Downloads & links

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Map

FileDownload / File: emd_13599.map.gz / Format: CCP4 / Size: 1.2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of mouse dorsal root ganglion axon. The volume was binned by 4 and deconvolved for visualization.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
11 Å/pix.
x 375 pix.
= 4125. Å		11 Å/pix.
x 927 pix.
= 10197. Å		11 Å/pix.
x 959 pix.
= 10549. 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: 11 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-5711.118 - 3524.2793
Average (Standard dev.)1.3318994e-06 (±389.3684)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions927959375
Spacing959927375
CellA: 10549.0 Å / B: 10197.0 Å / C: 4125.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111111
M x/y/z959927375
origin x/y/z0.0000.0000.000
length x/y/z10549.00010197.0004125.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS959927375
D min/max/mean-5711.1183524.2790.000

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Supplemental data

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Sample components

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Entire : Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset ...

EntireName: Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2 (TS_41).
Components
  • Cell: Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2 (TS_41).

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Supramolecule #1: Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset ...

SupramoleculeName: Tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2 (TS_41).
type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Example tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2. Tomogram was used for survey of microtubules and components in axons and for subtomogram average determination of ...Details: Example tomogram of a mouse dorsal root ganglion (DRG) axon from dataset 2. Tomogram was used for survey of microtubules and components in axons and for subtomogram average determination of microtubules and microtubule inner proteins.
Source (natural)Organism: Mus musculus (house mouse) / Organ: Neurons / Tissue: Dorsal root ganglion

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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: 7.4
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV / Details: Manual blot for 3 s before plunging.
DetailsAdult DRG neurons were grown for 7 days in vitro before vitrification. Tilt series was collected in a thin region of an axon.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI Solutions / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-10 / Average exposure time: 1.7 sec. / Average electron dose: 1.85 e/Å2
Details: 61 images per tilt series with 112.85 e/A2 total dose.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination 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: 53000
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

DetailsFrame alignment and dose-filtering was done with alignframes and newstack (IMOD) operated via subTOM. Tilt series alignment was performed in IMOD. Tomograms were reconstructed in novaCTF operated via subTOM.
Final reconstructionSoftware - Name: NOVACTF (ver. 1.0.0) / Number images used: 61
CTF correctionSoftware: (Name: NOVACTF (ver. 1.0.0), IMOD (ver. 4.10.32))
Details: Defoci were estimated with CTFPLOTTER (IMOD version 4.10.32). CTF correction was done with novaCTF.

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