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- EMDB-5885: Preparation of Primary Neurons for Visualizing Neurites in a Froz... -

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Basic information

Entry
Database: EMDB / ID: EMD-5885
TitlePreparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
Map dataReconstruction of a rat dorsal root ganglion neurite
Sample
  • Sample: Rat dorsal root ganglion neurite
  • Organelle or cellular component: dorsal root ganglion neurite
Keywordsneurites / neurobiology / brain / rat / primary neuron culture / hippocampal / dorsal root ganglion / cryo-electron tomography / cryo-electron microscopy
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / cryo EM
AuthorsShahmoradian SH / Galiano MR / Wu C / Chen S / Rasband MN / Mobley WC / Chiu W
CitationJournal: J Vis Exp / Year: 2014
Title: Preparation of primary neurons for visualizing neurites in a frozen-hydrated state using cryo-electron tomography.
Authors: Sarah H Shahmoradian / Mauricio R Galiano / Chengbiao Wu / Shurui Chen / Matthew N Rasband / William C Mobley / Wah Chiu /
Abstract: Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding ...Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.
History
DepositionJan 20, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseMar 19, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5885.map.gz / Format: CCP4 / Size: 222.7 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationReconstruction of a rat dorsal root ganglion neurite
Voxel sizeX=Y=Z: 21.16 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)6.1027317 (±14.800007819999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin11400
Dimensions22810241024
Spacing22810241024
CellA: 21667.84 Å / B: 4824.48 Å / C: 21667.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z21.1621.1621.16
M x/y/z10242281024
origin x/y/z0.0000.0000.000
length x/y/z21667.8404824.48021667.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS01140
NC/NR/NS10242281024
D min/max/mean-128.000127.0006.103

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Supplemental data

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Sample components

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Entire : Rat dorsal root ganglion neurite

EntireName: Rat dorsal root ganglion neurite
Components
  • Sample: Rat dorsal root ganglion neurite
  • Organelle or cellular component: dorsal root ganglion neurite

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Supramolecule #1000: Rat dorsal root ganglion neurite

SupramoleculeName: Rat dorsal root ganglion neurite / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: dorsal root ganglion neurite

SupramoleculeName: dorsal root ganglion neurite / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat / 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

VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.0 µm / Nominal defocus min: 7.0 µm / Nominal magnification: 20000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 5 °
DateOct 4, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Average electron dose: 60 e/Å2

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Image processing

Final reconstructionNumber images used: 1

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