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

Basic information

• Entry: Database: EMDB / ID: EMD-4490
• Title: cryo-ET of cryo-FIB milled HeLa cells overexpressing GFP-Bax
• Map data: Reconstructed cryo-tomogram of mitochondrion in HeLa cells overexpressing GFP-Bax

Sample

• Cell: HeLa (homo sapiens)

• Biological species: Homo sapiens (human)
• Method: electron tomography / cryo EM
• Authors: Ader NR / Hoffmann PC / Ganeva I / Borgeaud AC / Wang C / Youle RJ / Kukulski W
• Citation: Journal: 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 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).

History

Deposition	Dec 20, 2018	-
Header (metadata) release	Feb 13, 2019	-
Map release	Feb 13, 2019	-
Update	Feb 13, 2019	-
Current status	Feb 13, 2019	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_4490.map.gz / Format: CCP4 / Size: 1.8 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstructed cryo-tomogram of mitochondrion in HeLa cells overexpressing GFP-Bax
• Voxel size: X=Y=Z: 15.01 Å

Density

Minimum - Maximum	-711.339699999999993 - 498.797399999999982
Average (Standard dev.)	3.5066628 (±71.154730000000001)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 -255 Dimensions 928 960 551 Spacing 960 928 551
Cell	A: 14409.601 Å / B: 13929.28 Å / C: 8270.51 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	15.010001041667	15.01	15.01
M x/y/z	960	928	551
origin x/y/z	0.000	0.000	0.000
length x/y/z	14409.601	13929.280	8270.510
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	-255
NC/NR/NS	960	928	551
D min/max/mean	-711.340	498.797	3.507

Supplemental data

Sample components

Entire : HeLa (homo sapiens)

• Entire: Name: HeLa (homo sapiens)

Components

• Cell: HeLa (homo sapiens)

Supramolecule #1: HeLa (homo sapiens)

• Supramolecule: Name: 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)

Experimental details

Structure determination

• Method: cryo EM
• Processing: electron tomography
• Aggregation state: cell

Sample preparation

• Buffer: pH: 7.4 ; 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)
• Grid: Model: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
• Vitrification: Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER; Details: Grids were manually backside blotted using Whatman filter paper No. 1 and vitrified using a manual plunger..
• Details: HeLa cells were grown for 24 h on 200 mesh gold grids with a holey carbon film R2/2 (Quantifoil) in 6-well plates and transfected with 2000 ng hBax-C3-EGFP plasmid in presence of Q-VD-OPh. 16 hours after transfection, cells were stained with MitoTracker Deep Red, grids were manually backside blotted using Whatman filter paper No. 1 and vitrified using a manual plunger.
• Sectioning: Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 1 nA / Focused ion beam - Duration: 10800 sec. / Focused ion beam - Temperature: 83 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 200 nm; Focused ion beam - Details: Cells were cryo-FIB milled to prepare lamellae using a Scios DualBeam FIB/SEM (FEI) equipped with a Quorum cryo-stage (PP3010T), following the protocol described in Schaffer et al. (2015). In brief, grids were coated with an organic Pt compound using the gas injection system for either 8 s at 12 mm working distance or 30 s at 13 mm working distance from a stage tilt of 25 degrees. The stage was then tilted so that the grid was at a 10 degree angle towards the ion beam for all subsequent steps. The electron beam was used at 13 pA and 5-10 kV to locate cells, 2 kV for subsequent imaging. The ion beam was used at 30 kV and 10 pA for imaging. Rough milling was performed at 30 kV ion beam voltage, and subsequently the current was reduced from 0.5 nA to 0.3 nA until a lamella thickness of 5 um was reached, and further to 0.1 nA until 1 um lamella thickness. Fine milling to a final lamella thickness of approximately 200 nm was performed either at 30 kV and 30 pA, or 16 kV and 11 pA ion beam setting.. The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Scios DualBeam FIB/SEM. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
• 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.
• Image recording: Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Average electron dose: 1.1 e/Ų; Details: 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.
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Final reconstruction: Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: eTomo (ver. 4.10.20) / Details: 10 iterations / Number images used: 83