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Yorodumi- EMDB-5887: Preparation of Primary Neurons for Visualizing Neurites in a Froz... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5887 | |||||||||
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Title | Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography | |||||||||
Map data | Reconstruction of a rat hippocampal neurite | |||||||||
Sample |
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Keywords | neurites / neurobiology / brain / rat / primary neuron culture / hippocampal / dorsal root ganglion / cryo-electron tomography / cryo-electron microscopy | |||||||||
Biological species | Rattus norvegicus (Norway rat) | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Shahmoradian SH / Galiano MR / Wu C / Chen S / Rasband MN / Mobley WC / Chiu W | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5887.map.gz | 497.6 MB | EMDB map data format | |
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Header (meta data) | emd-5887-v30.xml emd-5887.xml | 7.6 KB 7.6 KB | Display Display | EMDB header |
Images | emd_5887.png | 776.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5887 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5887 | HTTPS FTP |
-Validation report
Summary document | emd_5887_validation.pdf.gz | 78 KB | Display | EMDB validaton report |
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Full document | emd_5887_full_validation.pdf.gz | 77.2 KB | Display | |
Data in XML | emd_5887_validation.xml.gz | 500 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5887 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5887 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5887.map.gz / Format: CCP4 / Size: 574.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of a rat hippocampal neurite | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 21.16 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Rat hippocampal neurite
Entire | Name: Rat hippocampal neurite |
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Components |
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-Supramolecule #1000: Rat hippocampal neurite
Supramolecule | Name: Rat hippocampal neurite / type: sample / ID: 1000 / Number unique components: 1 |
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-Supramolecule #1: hippocampal neurite
Supramolecule | Name: hippocampal neurite / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) / synonym: Rat / Tissue: Hippocampus |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Vitrification | Cryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK III |
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-Electron microscopy
Microscope | JEOL 2100 |
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Date | Jul 1, 2011 |
Image recording | Category: CCD / Film or detector model: GENERIC CCD / Average electron dose: 60 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 7.0 µm / Nominal defocus min: 7.0 µm / Nominal magnification: 20000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 5 ° |
-Image processing
Final reconstruction | Number images used: 1 |
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