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- EMDB-0995: Ultra-high voltage electron microscope tomography using 700-nm-th... -

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Entry
Database: EMDB / ID: EMD-0995
TitleUltra-high voltage electron microscope tomography using 700-nm-thick neurite section acquired at 15,000 magnification at an accelerating voltage of 1 MV
Map data700nm-thick neurite section of cultured PC12 cells (Magnification 15K)
Sample
  • Organelle or cellular component: Microtubule distribution in neurite of differentiated PC12 cell
Biological speciesRattus norvegicus (Norway rat)
Methodelectron tomography / negative staining
AuthorsNishida T / Yoshimura R / Nishi R / Imoto Y / Endo Y
CitationJournal: J Microsc / Year: 2020
Title: Application of ultra-high voltage electron microscope tomography to 3D imaging of microtubules in neurites of cultured PC12 cells.
Authors: T Nishida / R Yoshimura / R Nishi / Y Imoto / Y Endo /
Abstract: Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures ...Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three-dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra-high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three-dimensional array of microtubules in a 0.7-µm-thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7-µm-thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial-section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely-distributed cellular organelles such as microtubules for neurite outgrowth. LAY DESCRIPTION: An optimal 3D visualisation of microtubule cytoskeleton using ultra-high voltage electron microscopy tomography The ultra-high voltage electron microscope (UHVEM) is able to visualise a micrometre-thick specimen at nanoscale spatial resolution because of the high-energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7-µm-thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network.
History
DepositionFeb 4, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateMay 20, 2020-
Current statusMay 20, 2020Processing site: PDBj / Status: Released

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Structure visualization

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

emd_0995.png

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Map

FileDownload / File: emd_0995.map.gz / Format: CCP4 / Size: 174.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation700nm-thick neurite section of cultured PC12 cells (Magnification 15K)
Voxel sizeX=Y=Z: 45.2 Å
Density
Minimum - Maximum-18725 - 8072
Average (Standard dev.)629.7966 (±291.19156)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin2-22
Dimensions100893497
Spacing934100897
CellA: 42216.8 Å / B: 45561.6 Å / C: 4384.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z45.20000107066445.20000198412745.2
M x/y/z934100897
origin x/y/z0.0000.0000.000
length x/y/z42216.80145561.6024384.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-222
NC/NR/NS934100897
D min/max/mean-18725.0008072.000629.797

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Supplemental data

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Sample components

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Entire : Microtubule distribution in neurite of differentiated PC12 cell

EntireName: Microtubule distribution in neurite of differentiated PC12 cell
Components
  • Organelle or cellular component: Microtubule distribution in neurite of differentiated PC12 cell

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Supramolecule #1: Microtubule distribution in neurite of differentiated PC12 cell

SupramoleculeName: Microtubule distribution in neurite of differentiated PC12 cell
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.3
StainingType: POSITIVE / Material: Uranyl Acetate, Lead
Sugar embeddingMaterial: resin
GridMaterial: COPPER / Mesh: 50 / Support film - Material: FORMVAR
SectioningUltramicrotomy - Instrument: Reichert-Jung Ultracut E / Ultramicrotomy - Temperature: 300 K / Ultramicrotomy - Final thickness: 700 nm
Fiducial markerManufacturer: BBI Solutions / Diameter: 20 nm

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Electron microscopy

MicroscopeHITACHI H3000 UHVEM
Electron beamAcceleration voltage: 1000 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 15000
Sample stageSpecimen holder model: OTHER
Image recordingFilm or detector model: OTHER / Average electron dose: 13.0 e/Å2

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 67

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