[English] 日本語
Yorodumi
- EMDB-4492: cryo-ET of cryo-FIB milled Bax/Bak double knockout HCT116 cells s... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-4492
Titlecryo-ET of cryo-FIB milled Bax/Bak double knockout HCT116 cells stably expressing GFP-Bax
Map dataReconstructed electron cryo-tomogram of mitochondria in HeLa cells overexpression GFP-Bax
Sample
  • Cell: Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax
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 20, 2018-
Header (metadata) releaseFeb 13, 2019-
Map releaseFeb 13, 2019-
UpdateFeb 13, 2019-
Current statusFeb 13, 2019Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Supplemental images

emd_4492.png

Downloads & links

-
Map

FileDownload / File: emd_4492.map.gz / Format: CCP4 / Size: 2.2 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationReconstructed electron cryo-tomogram of mitochondria in HeLa cells overexpression GFP-Bax
Voxel sizeX=Y=Z: 7.506 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)1.4731983 (±23.165863000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-3133291
Dimensions18561920651
Spacing19201856651
CellA: 14411.5205 Å / B: 13931.136 Å / C: 4886.4062 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z7.50600052083337.5067.506
M x/y/z19201856651
origin x/y/z0.0000.0000.000
length x/y/z14411.52113931.1364886.406
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS33-31291
NC/NR/NS19201856651
D min/max/mean-128.000127.0001.473

-
Supplemental data

-
Sample components

-
Entire : Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax

EntireName: Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax
Components
  • Cell: Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax

-
Supramolecule #1: Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax

SupramoleculeName: Bax/Bak double knockout cell (homo sapiens) stably expressing GFP-Bax
type: cell / ID: 1 / Parent: 0
Details: Cryofixation of cell was performed 3 h after treatment with ABT-737
Source (natural)Organism: Homo sapiens (human)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

-
Sample preparation

BufferpH: 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)
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Grids were manually backside blotted using Whatman filter paper No. 1 and vitrified using a manual plunger..
DetailsBax/Bak DKO HCT116 GFP-Bax cells were grown on grids for 36 h, stained with MitoTracker Deep Red, and incubated with ABT-737 and Q-VD-OPh for 3 h before plunge-freezing. Grids were manually backside blotted using Whatman filter paper No. 1 and vitrified using a manual plunger.
SectioningFocused 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 ...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

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 either 190.9 or 99.4 nm. Intermediate magnification maps of lamella 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

-
Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: eTomo (ver. 4.10.20) / Details: 10 iterations / Number images used: 99

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more