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- EMDB-4483: Correlative FM and ET of GFP-Bax in HeLa cells -

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Basic information

Entry
Database: EMDB / ID: EMD-4483
TitleCorrelative FM and ET of GFP-Bax in HeLa cells
Map dataReconstructed tomogram of mitochondria in HeLa cells overexpressing GFP-Bax.
Sample
  • Cell: HeLa (homo sapiens)
Biological speciesHomo sapiens (human)
Methodelectron tomography / negative staining
AuthorsAder NR / Hoffmann PC / Ganeva I / Borgeaud AC / Wang C / Youle RJ / Kukulski W
CitationJournal: Elife / Year: 2019
Title: Molecular and topological reorganizations in mitochondrial architecture interplay during Bax-mediated steps of apoptosis.
Authors: Nicholas R Ader / Patrick C Hoffmann / Iva Ganeva / Alicia C Borgeaud / Chunxin Wang / Richard J Youle / Wanda Kukulski /
Abstract: During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing ...During apoptosis, Bcl-2 proteins such as Bax and Bak mediate the release of pro-apoptotic proteins from the mitochondria by clustering on the outer mitochondrial membrane and thereby permeabilizing it. However, it remains unclear how outer membrane openings form. Here, we combined different correlative microscopy and electron cryo-tomography approaches to visualize the effects of Bax activity on mitochondria in human cells. Our data show that Bax clusters localize near outer membrane ruptures of highly variable size. Bax clusters contain structural elements suggesting a higher order organization of their components. Furthermore, unfolding of inner membrane cristae is coupled to changes in the supramolecular assembly of ATP synthases, particularly pronounced at membrane segments exposed to the cytosol by ruptures. Based on our results, we propose a comprehensive model in which molecular reorganizations of the inner membrane and sequestration of outer membrane components into Bax clusters interplay in the formation of outer membrane ruptures.
EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all ...EDITORIAL NOTE: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
History
DepositionDec 19, 2018-
Header (metadata) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateFeb 13, 2019-
Current statusFeb 13, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • 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_4483.png

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Map

FileDownload / File: emd_4483.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationReconstructed tomogram of mitochondria in HeLa cells overexpressing GFP-Bax.
Voxel sizeX=Y=Z: 11.02 Å
Density
Minimum - Maximum-535.0 - 885.0
Average (Standard dev.)416.35205000000002 (±23.344325999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-80
Dimensions20482048160
Spacing20482048160
CellA: 22568.96 Å / B: 22568.96 Å / C: 1763.2001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z11.02000048828111.02000048828111.02
M x/y/z20482048160
origin x/y/z0.0000.0000.000
length x/y/z22568.96122568.9611763.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-80
NC/NR/NS20482048160
D min/max/mean-535.000885.000416.352

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Supplemental data

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Sample components

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Entire : HeLa (homo sapiens)

EntireName: HeLa (homo sapiens)
Components
  • Cell: HeLa (homo sapiens)

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Supramolecule #1: HeLa (homo sapiens)

SupramoleculeName: HeLa (homo sapiens) / type: cell / ID: 1 / Parent: 0
Details: Cryofixation of cell was performed 16 h after transfection of GFP-Bax plasmid.
Source (natural)Organism: Homo sapiens (human)

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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: 8.4 / Component - Name: PBS
StainingType: POSITIVE / Material: Uranyl Acetate / Details: 0.008% uranyl acetate in acetone
Sugar embeddingMaterial: 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 ...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.
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
High pressure freezingInstrument: 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 ...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.
SectioningUltramicrotomy - Instrument: Ultracut E Microtome (Reichert)
Ultramicrotomy - Temperature: 296 K / Ultramicrotomy - Final thickness: 300 nm
Fiducial markerManufacturer: Agar Scientific Ltd. / Diameter: 15 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: OTHER
DetailsET 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 recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Average electron dose: 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: ...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]
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo (ver. 4.10.20) / Details: 237 tilted images used from two axies / Number images used: 237

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