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- EMDB-12639: In situ subtomogram average of 13 protofilament microtubule from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-12639
TitleIn situ subtomogram average of 13 protofilament microtubule from Mus musculus DRG axons
Map datamicrotubule structure from mouse DRG axons
Sample
  • Organelle or cellular component: In situ subtomogram average of 13 protofilament microtubule from Mus musculus DRG axons
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 12.0 Å
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
DepositionMar 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
  • Surface view with section colored by density value
  • Surface level: 0.0719
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0719
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12639.png

Downloads & links

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Map

FileDownload / File: emd_12639.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmicrotubule structure from mouse DRG axons
Voxel sizeX=Y=Z: 2.75 Å
Density
Contour LevelBy AUTHOR: 0.0719 / Movie #1: 0.0719
Minimum - Maximum-0.24335557 - 0.33154163
Average (Standard dev.)-0.00021993526 (±0.063600354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 528.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.752.752.75
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z528.000528.000528.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2430.332-0.000

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Supplemental data

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Mask #1

Fileemd_12639_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12639_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12639_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : In situ subtomogram average of 13 protofilament microtubule from ...

EntireName: In situ subtomogram average of 13 protofilament microtubule from Mus musculus DRG axons
Components
  • Organelle or cellular component: In situ subtomogram average of 13 protofilament microtubule from Mus musculus DRG axons

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Supramolecule #1: In situ subtomogram average of 13 protofilament microtubule from ...

SupramoleculeName: In situ subtomogram average of 13 protofilament microtubule from Mus musculus DRG axons
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse) / Organ: Neurons / Tissue: Dorsal root ganglion / Organelle: microtubule cytoskeleton / Location in cell: axoplasm

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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: 7.4
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
Details: Grids were additionally coated in 0.1mg/mL poly-L-lysine then 0.01mg/mL laminin before cell plating
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV / Details: Manual blot for 3 s before plunging.
DetailsMicrotubules in axons of adult DRG neurons grown for 7 days in vitro

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 53000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 39 / Number images used: 476268 / Reference model: average of all particles (tube-shape) / Method: Dynamo filament extraction
Software:
Namedetails
IMOD (ver. 4.10.32)model
Dynamo (ver. 1.1.333)particle position determination

Details: Paths of microtubules were modelled in IMOD (version 4.10.32). After importing into Dynamo, initial particle positions were determined using the 'filamentWithTorsion' model workflow. Helical ...Details: Paths of microtubules were modelled in IMOD (version 4.10.32). After importing into Dynamo, initial particle positions were determined using the 'filamentWithTorsion' model workflow. Helical symmetry was applied at the particle level and subvolumes were extracted using subTOM.
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.
Final angle assignmentType: OTHER
Details: Alignment, classification and averaging was performed in subTOM (1.1.5)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Software - details: postprocess
Details: Particles were aligned together at bin4. Half maps were generated at bin4 and independently aligned at bin2 and bin1.
Number subtomograms used: 64528
FSC plot (resolution estimation)

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