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- EMDB-12640: In situ subtomogram average of microtubule inner protein from Mus... -

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Basic information

Entry
Database: EMDB / ID: EMD-12640
TitleIn situ subtomogram average of microtubule inner protein from Mus musculus DRG axons
Map datamicrotubule inner protein structure from mouse DRG axon microtubules
Sample
  • Organelle or cellular component: In situ subtomogram average of microtubule inner protein from Mus musculus DRG axons
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 32.0 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: 0.085
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.085
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12640.png

Downloads & links

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Map

FileDownload / File: emd_12640.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmicrotubule inner protein structure from mouse DRG axon microtubules
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.5 Å/pix.
x 64 pix.
= 352. Å		5.5 Å/pix.
x 64 pix.
= 352. Å		5.5 Å/pix.
x 64 pix.
= 352. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 5.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.085 / Movie #1: 0.085
Minimum - Maximum-0.3071782 - 0.56908214
Average (Standard dev.)0.0012990833 (±0.033401303)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.55.55.5
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.3070.5690.001

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Supplemental data

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Mask #1

Fileemd_12640_msk_1.map
Projections & Slices
AxesZYX

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Half map: half map1

Fileemd_12640_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

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Half map: half map2

Fileemd_12640_half_map_2.map
Annotationhalf map2
Projections & Slices
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Sample components

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Entire : In situ subtomogram average of microtubule inner protein from Mus...

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

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Supramolecule #1: In situ subtomogram average of microtubule inner protein from Mus...

SupramoleculeName: In situ subtomogram average of microtubule inner protein 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 plating
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV / Details: Manual blot for 3 s before plunging.
DetailsMicrotubule inner proteins in axons of adult DRG neurons grown for 7 days in vitro

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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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Software - details: postprocess
Details: Half maps were generated in subTOM and then postprocessed in Relion. The final reconstruction was lowpass filtered to 32A.
Number subtomograms used: 3286
ExtractionNumber tomograms: 10 / Number images used: 3852
Software: (Name: IMOD (ver. 4.10.32), Dynamo (ver. 1.1.333))
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)
FSC plot (resolution estimation)

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