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Yorodumi- EMDB-2186: Dual-axis cryo-electron tomogram of microtubules assembled in vitro -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2186 | |||||||||
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Title | Dual-axis cryo-electron tomogram of microtubules assembled in vitro | |||||||||
Map data | Dual-axis cryo-electron tomogram of microtubules assembled in vitro | |||||||||
Sample |
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Keywords | microtubules / single-axis / dual-axis / cryo-electron tomography / missing wedge | |||||||||
Biological species | Sus scrofa (pig) | |||||||||
Method | electron tomography / cryo EM / Resolution: 40.0 Å | |||||||||
Authors | Guesdon A / Blestel S / Kervrann C / Chretien D | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2186.map.gz | 250.8 MB | EMDB map data format | |
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Header (meta data) | emd-2186-v30.xml emd-2186.xml | 10 KB 10 KB | Display Display | EMDB header |
Images | EMD-2186.tif | 244.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2186 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2186 | HTTPS FTP |
-Validation report
Summary document | emd_2186_validation.pdf.gz | 297.1 KB | Display | EMDB validaton report |
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Full document | emd_2186_full_validation.pdf.gz | 296.2 KB | Display | |
Data in XML | emd_2186_validation.xml.gz | 4.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2186 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2186 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2186.map.gz / Format: CCP4 / Size: 293 MB / Type: IMAGE STORED AS SIGNED BYTE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Dual-axis cryo-electron tomogram of microtubules assembled in vitro | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.5 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 : Cryo-electron tomogram of microtubules
Entire | Name: Cryo-electron tomogram of microtubules |
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Components |
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-Supramolecule #1000: Cryo-electron tomogram of microtubules
Supramolecule | Name: 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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-Macromolecule #1: Microtubule
Macromolecule | Name: Microtubule / type: protein_or_peptide / ID: 1 / Details: Polymer of the tubulin molecule / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
-Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 6.9 / Details: 80mM PIPES, 1mM EGTA, 1mM MgCl2, 50mM KCl, 1mM GTP |
Grid | Details: 300 mesh homemade holey-carbon grids, glow discharged |
Vitrification | Cryogen 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 |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Average: 92 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Details | Defocus value is nominal. Electron dose is per image. Second axis tilt angles: -50.47 to 62.56 degrees. |
Date | Oct 21, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 75 / Average electron dose: 0.3 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 32563 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus min: 3.0 µm / Nominal magnification: 25000 |
Sample stage | Specimen holder: Model CT3500TR from Gatan, nominal tilt angle +/- 60 degrees, in-plane rotation 120 degrees. Cooled by liquid nitrogen. Specimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -57.74 ° / Tilt series - Axis1 - Max angle: 55.94 ° / Tilt series - Axis1 - Angle increment: 3.7 ° |
-Image processing
Details | Dual-axis acquisition: first axis 37 projections, second axis 38 projections The in-plane rotation angle between the two series is 93.5 degrees The overlap between the two series is 78 percent We used a Saxton acquisition scheme for tilt increment Gold beads used as fiducial markers were erased |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: Imod Details: Logarithm of densities offset: 32768 Tomogram thickness in z: 300 X-axis tilt, first axis: 2.8, second axis: 1.28 Radial filter cutoff: 0.1 Radial filter falloff: 0.1 Use Z factors Number images used: 75 |