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- EMDB-4486: Correlative FM and cryo-ET of GFP-Bax in HeLa cells -

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Basic information

Entry
Database: EMDB / ID: EMD-4486
TitleCorrelative FM and cryo-ET of GFP-Bax in HeLa cells
Map dataReconstructed electron cryo-tomogram of HeLa cell overexpressing GFP-Bax
Sample
  • Cell: HeLa (homo sapiens)
Biological speciesHomo 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 ...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

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Map

FileDownload / File: emd_4486.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationReconstructed electron cryo-tomogram of HeLa cell overexpressing GFP-Bax
Voxel sizeX=Y=Z: 7.534 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-5.343136 (±10.356811)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-189
Dimensions18541920380
Spacing19201854380
CellA: 14465.28 Å / B: 13968.036 Å / C: 2862.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z7.5347.5347.534
M x/y/z19201854380
origin x/y/z0.0000.0000.000
length x/y/z14465.28013968.0362862.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-189
NC/NR/NS19201854380
D min/max/mean-128.000127.000-5.343

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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

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Component - Name: PBS
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)
GridModel: Quantifoil R3.5/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: NITROGEN / Details: high pressure frozen.
High pressure freezingInstrument: OTHER
Details: HeLa cells were grown for 24 h in 6-well plates, transfected with 2000 ng hBax-C3-EGFP plasmid and incubated with Q-VD-OPh for 16 h, then trypsinized and pelleted. Immediately before ...Details: HeLa cells were grown for 24 h in 6-well plates, transfected with 2000 ng hBax-C3-EGFP plasmid and incubated with Q-VD-OPh for 16 h, then trypsinized and pelleted. Immediately before trypsinizing, cells were stained with MitoTracker Deep Red. Pellets were maintained at 37 degrees C while they were mixed 1:1 with 40% Dextran (Sigma) in PBS, pipetted into the 0.2 mm recess of gold-coated copper carriers, covered with the flat side of Aluminum carriers B and high-pressure frozen with a Leica HPM100 (Leica Microsystems).. 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.
Cryo protectant40% Dextran
SectioningUltramicrotomy - Instrument: Leica UC6/FC6 / Ultramicrotomy - Temperature: 123 K / Ultramicrotomy - Final thickness: 100 nm
Ultramicrotomy - Details: 100 nm thick vitreous sections were produced at -150 degrees C in a UC6/FC6 cryo-ultramicrotome (Leica Microsystems) using cryotrim 25 and a 35 degree cryo immuno knives ...Ultramicrotomy - Details: 100 nm thick vitreous sections were produced at -150 degrees C in a UC6/FC6 cryo-ultramicrotome (Leica Microsystems) using cryotrim 25 and a 35 degree cryo immuno knives (Diatome). The sections were attached using a Crion antistatic device (Leica Microsystems) to EM grids (R3.5/1, copper, Quantifoil) that were plasma cleaned and had 100 nm TetraSpeck beads (Invitrogen) diluted 1:50 in PBS adhered to them.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 5.0 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsMontaged images of the entire grid were acquired at low magnification at pixel size of 182.3 nm. Intermediate magnification maps of grid squares with vitreous sections were acquired at pixel size 5.5 nm.
Image recordingFilm 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/Å2
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 ...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.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: eTomo (ver. 4.10.20)
Details: NUmber of tilted images used in for this volume is approximate.
Number images used: 70

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