EMD-3558
Centriolar Proximal MT triplet in centrosomes extracted from o.aries thymocytes
EMD-3558
Subtomogram averaging47.0 Å
Deposition: 24/12/2016Map released: 22/08/2018
Last modified: 22/08/2018
Concentration: 0.076
mg/mL
Details: The sample was enriched by two step of sedimentation in sucrose gradient. The sucrose was washed out in several drops of buffer, the last of which included fiducial markers (10 nm bovine serum albumin (BSA)-coated gold beads) for tilt series alignment
Details: The sample was enriched by two step of sedimentation in sucrose gradient. The sucrose was washed out in several drops of buffer, the last of which included fiducial markers (10 nm bovine serum albumin (BSA)-coated gold beads) for tilt series alignment
Buffer
pH: 7.2
Buffer components [1]:
Details: PH rectification with KOH
Buffer components [1]:
| Name | Formula | Concentration | ChEBI |
|---|---|---|---|
| Pipes | C8H18N2O6S2 | 10.0 mM |
Grid
Vitrification
Microscope: FEI TITAN KRIOS
Illumination mode: OTHER
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 300 kV
Nominal CS: 2.0 mm
Nominal defocus: 5.0 µm - 5.0 µm
Calibrated defocus: 5.54 µm - 5.906 µm
Nominal magnification: 42000.0
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cooling holder cryogen: NITROGEN
Illumination mode: OTHER
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 300 kV
Nominal CS: 2.0 mm
Nominal defocus: 5.0 µm - 5.0 µm
Calibrated defocus: 5.54 µm - 5.906 µm
Nominal magnification: 42000.0
Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Cooling holder cryogen: NITROGEN
Specialist optics
Energy filter
Image Recording
[1]
Detector model:
GATAN K2 QUANTUM (4k x 4k)
Dimensions: 3838 pixel x 3710 pixel
Average exposure time: 1.0 s
Average electron dose per image: 1.4 e/Å2
Dimensions: 3838 pixel x 3710 pixel
Average exposure time: 1.0 s
Average electron dose per image: 1.4 e/Å2
Details: Tilt series were aligned based on fiducials with IMOD and mass normalized using PRIISM. The ctf correction was processed on tilt series using TomoCTF. Tomogram were reconstructed using SIRT algorithm with TOMO3D. The number of frames per image were variable, with 8 frames at full tilt, through to 4 frames at zero tilt.
Final
reconstruction
Resolution: 47.0
Å
(
BY AUTHOR)
Resolution method: FSC 0.5 CUT-OFF
Number of classed used: 2
Algorithm: FOURIER SPACE
Details:
Resolution method: FSC 0.5 CUT-OFF
Number of classed used: 2
Algorithm: FOURIER SPACE
Details:
Software
[1]
| Name | Version | Details |
|---|---|---|
| Dynamo | 1.1.22 | - |
Crystal parameters
⦩ Final angle assignment
Type:
OTHER
Details: The particular geometry of the centriole with its repeat every 8.3nm along the proximal-distal axis and its coarse 9 fold symmetry was used to construct from each tomogram a model lowly affected by missing wedge. Those coarse models were volume aligned and an initial reference were averaged from all the subtomogram particle using the coarse orientation of the particles determined that way. This model was iteratively refined. The reference model for the next iteration was made using 70% of the particles based on the best correlation during the alignment. For the finer step of refinement, a mask was used to restrict the alignment to an area covering 3 of the 8.3nm repeats.
Details: The particular geometry of the centriole with its repeat every 8.3nm along the proximal-distal axis and its coarse 9 fold symmetry was used to construct from each tomogram a model lowly affected by missing wedge. Those coarse models were volume aligned and an initial reference were averaged from all the subtomogram particle using the coarse orientation of the particles determined that way. This model was iteratively refined. The reference model for the next iteration was made using 70% of the particles based on the best correlation during the alignment. For the finer step of refinement, a mask was used to restrict the alignment to an area covering 3 of the 8.3nm repeats.
Software
[1]
| Name | Version | Details |
|---|---|---|
| Dynamo | 1.1.22 | - |
Final 3D classification
Number of classes:
4
Details: Classification by PCA + k-means
Details: Classification by PCA + k-means
Software
[1]
| Name | Version | Details |
|---|---|---|
| Dynamo | 1.1.22 | - |
Extraction
Number of images used: 800
Method: Volume picked interactively
Method: Volume picked interactively
Software
[1]
| Name | Version | Details |
|---|---|---|
| Dynamo | 1.1.22 | - |
CTF correction
Details: Wiener filtering, made on tilt series
Software
[1]
| Name | Version | Details |
|---|---|---|
| TOMOCTF | - | - |
Format: CCP4
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: Centriolar proximal MT triplet from a mammal centrosome. Centrosomes were extracted from o.aries thymocytes.
Data type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation details: Centriolar proximal MT triplet from a mammal centrosome. Centrosomes were extracted from o.aries thymocytes.
⬡ Geometry
| X | Y | Z | |
|---|---|---|---|
| Origin | 0 | 0 | 0 |
| Dimensions (px) | 300 | 300 | 300 |
| Dimensions (Å) | 1026.0 | 1026.0 | 1026.0 |
| Voxel size (Å) | 3.42 | 3.42 | 3.42 |
Contour list
| Primary | Level | Source |
|---|---|---|
| True | 0.4 | AUTHOR |