+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4486 | |||||||||
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Title | Correlative FM and cryo-ET of GFP-Bax in HeLa cells | |||||||||
Map data | Reconstructed electron cryo-tomogram of HeLa cell overexpressing GFP-Bax | |||||||||
Sample |
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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 ...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 |
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-Structure visualization
Movie |
Movie viewer |
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Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4486.map.gz | 894 MB | EMDB map data format | |
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Header (meta data) | emd-4486-v30.xml emd-4486.xml | 12.5 KB 12.5 KB | Display Display | EMDB header |
Images | emd_4486.png | 139.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4486 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4486 | HTTPS FTP |
-Validation report
Summary document | emd_4486_validation.pdf.gz | 163.2 KB | Display | EMDB validaton report |
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Full document | emd_4486_full_validation.pdf.gz | 162.3 KB | Display | |
Data in XML | emd_4486_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4486 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4486 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_4486.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS SIGNED BYTE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstructed electron cryo-tomogram of HeLa cell overexpressing GFP-Bax | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 7.534 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : HeLa (homo sapiens)
Entire | Name: HeLa (homo sapiens) |
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Components |
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-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. |
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Source (natural) | Organism: Homo sapiens (human) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 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) |
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Grid | Model: Quantifoil R3.5/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING |
Vitrification | Cryogen name: NITROGEN / Details: high pressure frozen. |
High pressure freezing | Instrument: 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 protectant | 40% Dextran |
Sectioning | Ultramicrotomy - 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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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 182.3 nm. Intermediate magnification maps of grid squares with vitreous sections 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/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. |
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: NUmber of tilted images used in for this volume is approximate. Number images used: 70 |
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