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- EMDB-2185: Single-axis cryo-electron tomogram of microtubules assembled in vitro -

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Basic information

Entry
Database: EMDB / ID: EMD-2185
TitleSingle-axis cryo-electron tomogram of microtubules assembled in vitro
Map datasingle-axis cryo-electron tomogram of microtubules assembled in vitro
Sample
  • Sample: Cryo-electron tomogram of microtubules
  • Organelle or cellular component: Microtubule
Keywordsmicrotubules / single-axis / dual-axis / cryo-electron tomography / missing wedge
Biological speciesSus scrofa (pig)
Methodelectron tomography / cryo EM / Resolution: 40.0 Å
AuthorsGuesdon A / Blestel S / Kervrann C / Chretien D
CitationJournal: J Struct Biol / Year: 2013
Title: Single versus dual-axis cryo-electron tomography of microtubules assembled in vitro: limits and perspectives.
Authors: Audrey Guesdon / Sophie Blestel / Charles Kervrann / Denis Chrétien /
Abstract: Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from ...Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. Here, we asked how a dual-axis acquisition scheme would improve three-dimensional reconstructions of microtubules assembled in vitro. We show that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. Analysis of their three-dimensional Fourier transform indicates that this imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells.
History
DepositionSep 6, 2012-
Header (metadata) releaseOct 10, 2012-
Map releaseDec 5, 2012-
UpdateFeb 13, 2013-
Current statusFeb 13, 2013Processing site: PDBe / Status: Released

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Structure visualization

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

emd-2185.tif

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Map

FileDownload / File: emd_2185.map.gz / Format: CCP4 / Size: 293 MB / Type: IMAGE STORED AS SIGNED BYTE
Annotationsingle-axis cryo-electron tomogram of microtubules assembled in vitro
Voxel sizeX=Y=Z: 8.5 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-27.270030980000001 (±105.208663939999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0018
Dimensions10241024300
Spacing10241024300
CellA: 8704.0 Å / B: 8704.0 Å / C: 2550.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z8.58.58.5
M x/y/z10241024300
origin x/y/z0.0000.0000.000
length x/y/z8704.0008704.0002550.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS0018
NC/NR/NS10241024300
D min/max/mean-128.000127.000-27.270

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Supplemental data

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Sample components

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Entire : Cryo-electron tomogram of microtubules

EntireName: Cryo-electron tomogram of microtubules
Components
  • Sample: Cryo-electron tomogram of microtubules
  • Organelle or cellular component: Microtubule

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Supramolecule #1000: Cryo-electron tomogram of microtubules

SupramoleculeName: Cryo-electron tomogram of microtubules / type: sample / ID: 1000
Details: Microtubules were polymerized from purified porcine brain tubulin
Number unique components: 1

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Supramolecule #1: Microtubule

SupramoleculeName: Microtubule / type: organelle_or_cellular_component / ID: 1 / Details: Polymer of the tubulin molecule / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 6.9 / Details: 80mM Pipes, 1mM EGTA, 1mM MgCl2,50mM KCl,1mM GTP
GridDetails: 300 mesh homemade holey-carbon grids, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: The incubation was performed in saturated humidity and warm (35 degrees) atmosphere.
Timed resolved state: Incubation 3 min on the grid before vitrification.
Method: 4 microliters of specimen were incubated on the grid at 35 degrees, and then blotted for ~2 sec before plunging

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 32563 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus min: 3.0 µm / Nominal magnification: 25000
Sample stageSpecimen holder: Model 626DH from gatan, nominal tilt angle +/- 70 degrees. Cooled by liquid nitrogen.
Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -62.40 ° / Tilt series - Axis1 - Max angle: 67.49 ° / Tilt series - Axis1 - Angle increment: 2.4 °
TemperatureAverage: 98 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DetailsDefocus value is nominal. Electron dose is per image.
DateMar 7, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 73 / Average electron dose: 0.3 e/Å2

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: Imod
Details: Logarithm of densities offset: 32768.0 Tomogram thickness in Z: 300 X-axis tilt: -1.02 Radial filter cutoff: 0.1 Radial filter Falloff: 0.1 Use Z factors
Number images used: 73
DetailsWe used a Saxton acquisition scheme for tilt increment Gold beads used as fiducial markers were erased

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