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- EMDB-10378: Cryo-electron tomogram of cER-PM MCS in a heat-shocked WT S. cere... -

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Basic information

Entry
Database: EMDB / ID: EMD-10378
TitleCryo-electron tomogram of cER-PM MCS in a heat-shocked WT S. cerevisiae cell
Map data
Sample
  • Cell: ER-PM membrane contact site
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsCollado J / Fernandez-Busnadiego R
Funding support United Kingdom, Switzerland, Germany, Spain, 11 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UU_00012/6 United Kingdom
Swiss National Science Foundation31003A_179517 Switzerland
European Research CouncilERC-2012-SyG_318987-ToPAG Germany
German Research FoundationEXC 2067/1- 390729940 Germany
European Research CouncilERC AdG TENDO - 834394 Switzerland
Spanish Ministry of Economy and CompetitivenessSEV-2015-0522 Spain
German Research FoundationSFB1190/P22 Germany
Spanish Ministry of Economy and CompetitivenessFIS2017-89560-R Spain
Spanish Ministry of Economy and CompetitivenessFIS2015-63550-R Spain
Spanish Ministry of Economy and CompetitivenessRYC-2017-22227 Spain
Spanish Ministry of Economy and CompetitivenessBFU2015-73288-JIN Spain
CitationJournal: Dev Cell / Year: 2019
Title: Tricalbin-Mediated Contact Sites Control ER Curvature to Maintain Plasma Membrane Integrity.
Authors: Javier Collado / Maria Kalemanov / Felix Campelo / Clélia Bourgoint / Ffion Thomas / Robbie Loewith / Antonio Martínez-Sánchez / Wolfgang Baumeister / Christopher J Stefan / Rubén Fernández-Busnadiego /
Abstract: Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in Saccharomyces ...Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in Saccharomyces cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22, and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here, we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. Notably, Tcb proteins (tricalbins) form peaks of extreme curvature on the cER membrane facing the PM. Combined modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids between the cER and the PM, which is necessary to maintain PM integrity under heat stress. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to regulate MCS function.
History
DepositionOct 15, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 13, 2019-
UpdateJun 3, 2020-
Current statusJun 3, 2020Processing 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

emd_10378.png

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Map

FileDownload / File: emd_10378.map.gz / Format: CCP4 / Size: 310.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-154 - 83
Average (Standard dev.)2.817814 (±11.099517)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin9400
Dimensions928928189
Spacing928928189
CellA: 12695.04 Å / B: 12695.04 Å / C: 2585.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928189
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0402585.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS0940
NC/NR/NS928928189
D min/max/mean-154.00083.0002.818

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Supplemental data

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Sample components

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Entire : ER-PM membrane contact site

EntireName: ER-PM membrane contact site
Components
  • Cell: ER-PM membrane contact site

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Supramolecule #1: ER-PM membrane contact site

SupramoleculeName: ER-PM membrane contact site / type: cell / ID: 1 / Parent: 0 / Details: Yeast cells vitrified after heat shock
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SEY6210.1

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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: 5.5 / Details: YPD medium
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 21 % / Chamber temperature: 300 K / Instrument: FEI VITROBOT MARK IV
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 7200 sec. / Focused ion beam - Temperature: 90 K / Focused ion beam - Initial thickness: 4000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is FEI Quanta FIB. This is not in a list of allowed values set(['DB235', 'OTHER']) so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Average exposure time: 1.8 sec. / Average electron dose: 1.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.9) / Details: Tomogram was binned twice. / Number images used: 55

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