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- EMDB-4490: cryo-ET of cryo-FIB milled HeLa cells overexpressing GFP-Bax -

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Entry
Database: EMDB / ID: 4490
Titlecryo-ET of cryo-FIB milled HeLa cells overexpressing GFP-Bax
Map dataReconstructed cryo-tomogram of mitochondrion in HeLa cells overexpressing GFP-Bax
SampleHeLa (homo sapiens):
SourceHomo sapiens (human)
Methodelectron tomography / cryo EM
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 the issues have been addressed (see decision letter).
DateDeposition: Dec 20, 2018 / Header (metadata) release: Feb 13, 2019 / Map release: Feb 13, 2019 / Last update: Feb 13, 2019

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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

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Map

Fileemd_4490.map.gz (map file in CCP4 format, 1963500 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
551 pix
15.01 Å/pix.
= 8270.51 Å
928 pix
15.01 Å/pix.
= 13929.28 Å
960 pix
15.01 Å/pix.
= 14409.601 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

(generated in cubic-lattice coordinate)

Voxel sizeX=Y=Z: 15.01 Å
Density
Minimum - Maximum-711.339699999999993 - 498.797399999999982
Average (Standard dev.)3.5066628 (71.154730000000001)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions928960551
Origin0.00.0-255.0
Limit927.0959.0295.0
Spacing960928551
CellA: 14409.601 Å / B: 13929.28 Å / C: 8270.51 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z15.01000104166715.0115.01
M x/y/z960928551
origin x/y/z0.0000.0000.000
length x/y/z14409.60113929.2808270.510
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS00-255
NC/NR/NS960928551
D min/max/mean-711.340498.7973.507

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Supplemental data

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Sample components

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Entire HeLa (homo sapiens)

EntireName: HeLa (homo sapiens)
Details: Cryofixation of cell was performed 16 h after transfection of GFP-Bax plasmid.
Number of components: 1

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Component #1: cellular-component, HeLa (homo sapiens)

Cellular-componentName: HeLa (homo sapiens)
Details: Cryofixation of cell was performed 16 h after transfection of GFP-Bax plasmid.
Recombinant expression: No
SourceSpecies: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionBuffer solution: DMEM, high glucose, GlutaMAX, pyruvate (Thermo 31996) medium supplemented with 10% heat-inactivated FBS (Gibco 10270), 10 mM HEPES, and 1x NEAA (Thermo 11140)
pH: 7.4
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Grids were manually backside blotted using Whatman filter paper No. 1 and vitrified using a manual plunger..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Montaged images of the entire grid were acquired at low magnification at pixel size of either 190.9 or 99.4 nm. Intermediate magnification maps of lamella were acquired at pixel size 5.5 nm.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Defocus: 5000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionDetails: Electron cryo-tomographic tilt-series were collected on a Titan Krios (FEI) operated at 300 kV using a Quantum energy filter (slit width 20 eV) and a K2 direct electron detector (Gatan) in counting mode at a pixel size of 3.7 angstroms and at a dose rate of ~ 2-4 e-/pixel/second on the detector, dependent on sample thickness. Tilt-series were acquired between +/- 60 degrees starting from 0 degrees with 1 degrees increment using SerialEM (Mastronarde, 2005) following a grouped dose-symmetric acquisition with a group size of 4 (Bharat et al., 2018; Hagen et al., 2017), and at -5 um defocus. A dose of approximately 1.0 to 1.2 e-/square angstroms was applied per image of the tilt-series.

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Image processing

ProcessingMethod: electron tomography / Number of sections: 83
3D reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software: eTomo / Details: 10 iterations

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