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EMD-4484
Correlative FM and ET of GFP-Bax in HCT116 cells
EMD-4484
Tomography
Deposition: 19/12/2018Map released: 13/02/2019
Last modified: 13/02/2019
Buffer
pH: 7.4
Buffer components [1]:
Details: DMEM, high glucose, GlutaMAX, pyruvate (Thermo 31996) medium supplemented with 10% heat-inactivated FBS (Gibco 10270), 10 mM HEPES, and 1x NEAA (Thermo 11140)
Buffer components [1]:
| Name | Formula | Concentration | ChEBI |
|---|---|---|---|
| DMEM | - | - | - |
Staining
Sugar
embedding
Material: Lowicryl HM20
Details: We use a temperature-controlling Leica AFS2 with FSP. FS is performed at -90 degrees C for 24-36 h in 0.008 percent (w/v) uranyl acetate in glass-distilled acetone. The temperature is then increased to -45 C (5 degrees C/h). Next, the samples are washed three times with acetone and infiltrated with increasing concentrations (10 percent , 25 percent, 50 percent, 75 percent, 2 h each) of Lowicryl HM20 in acetone. During the final mix, the temperature is raised to -35 degrees C (2.5 degree C/h). The temperature is then raised further to -25 degrees C (2.5 degrees C/h), while 100 percent Lowicryl is exchanged three times in 4 h steps with agitation. Then, UV light is applied for 24 h to initialize Lowicryl polymerization. The temperature is then raised to 20 degrees C (5 degrees C/h). At this point, samples can be taken out of the AFS2, but we wait at least 2 days before removing blocks from the plastic wheel to ensure complete polymerization.
Details: We use a temperature-controlling Leica AFS2 with FSP. FS is performed at -90 degrees C for 24-36 h in 0.008 percent (w/v) uranyl acetate in glass-distilled acetone. The temperature is then increased to -45 C (5 degrees C/h). Next, the samples are washed three times with acetone and infiltrated with increasing concentrations (10 percent , 25 percent, 50 percent, 75 percent, 2 h each) of Lowicryl HM20 in acetone. During the final mix, the temperature is raised to -35 degrees C (2.5 degree C/h). The temperature is then raised further to -25 degrees C (2.5 degrees C/h), while 100 percent Lowicryl is exchanged three times in 4 h steps with agitation. Then, UV light is applied for 24 h to initialize Lowicryl polymerization. The temperature is then raised to 20 degrees C (5 degrees C/h). At this point, samples can be taken out of the AFS2, but we wait at least 2 days before removing blocks from the plastic wheel to ensure complete polymerization.
Grid
High
pressure freezing
Instrument:
OTHER
Details: HPF is accomplished using a Leica HPM100 in the provided temperature- and humidity-controlled chamber. To high-pressure freeze cells, we use a carrier method described in the manual for the Leica EM HPM100 CLEM 3 mm system for HPF of sapphire disks. In brief, carriers are assembled as follows between two plastic half cylinders: (1) 6 mm copper gold-plated support ring in a 6 mm CLEM middle plate, (2) a 3 mm sapphire (cells up), (3) a 3 mm spacer ring, (4) a clean sapphire, and (5) a 6 mm cover ring.. The value given for _emd_high_pressure_freezing.instrument is Leica EM HP100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
Details: HPF is accomplished using a Leica HPM100 in the provided temperature- and humidity-controlled chamber. To high-pressure freeze cells, we use a carrier method described in the manual for the Leica EM HPM100 CLEM 3 mm system for HPF of sapphire disks. In brief, carriers are assembled as follows between two plastic half cylinders: (1) 6 mm copper gold-plated support ring in a 6 mm CLEM middle plate, (2) a 3 mm sapphire (cells up), (3) a 3 mm spacer ring, (4) a clean sapphire, and (5) a 6 mm cover ring.. The value given for _emd_high_pressure_freezing.instrument is Leica EM HP100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
Fiducial Markers
| Manufacturer | Fiducial type | Diameter |
|---|---|---|
| Agar Scientific Ltd. | - | 20 nanometer |
Sectioning (Ultramicrotomy)
Microscope: FEI TECNAI F20
Illumination mode: SPOT SCAN
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 200 kV
C2 aperture diameter: 50.0 µm
Specimen holder model: OTHER
Alignment procedure: BASIC
Details: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm.
Illumination mode: SPOT SCAN
Imaging mode: BRIGHT FIELD
Electron source: FIELD EMISSION GUN
Acceleration voltage: 200 kV
C2 aperture diameter: 50.0 µm
Specimen holder model: OTHER
Alignment procedure: BASIC
Details: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm.
Image Recording
[1]
Detector model:
OTHER
Dimensions: 2048 pixel x 2048 pixel
Average electron dose per image: 1000.0 e/Å2
Details: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm. [NOTE: Electron dose unknown. Value specified here is a dummy]
Dimensions: 2048 pixel x 2048 pixel
Average electron dose per image: 1000.0 e/Å2
Details: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder (Model 2020; Fischione Instruments) at a 1.1nm pixel size with a camera length of 200 mm. [NOTE: Electron dose unknown. Value specified here is a dummy]
Final
reconstruction
Number of images used:
222
Algorithm: BACK PROJECTION
Algorithm: BACK PROJECTION
Software
[1]
| Name | Version | Details |
|---|---|---|
| ETomo | 4.10.20 | - |
Format: CCP4
Data type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation details: Reconstructed tomogram of mitochondria in Bax/Bak double knockout HCT116 cells stably expressing GFP-Bax following treatment with ABT-737.
Data type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Annotation details: Reconstructed tomogram of mitochondria in Bax/Bak double knockout HCT116 cells stably expressing GFP-Bax following treatment with ABT-737.
⬡ Geometry
| X | Y | Z | |
|---|---|---|---|
| Origin | 0 | 0 | -88 |
| Dimensions (px) | 2048 | 2048 | 176 |
| Dimensions (Å) | 22568.96 | 22568.96 | 1939.52 |
| Voxel size (Å) | 11.02 | 11.02 | 11.02 |
Contour list
| Primary | Level | Source |
|---|---|---|
| True | - | AUTHOR |