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- EMDB-10309: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10309
Titlecryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are deleted, with high intracellular calcium; shown in Figures 5C and S3F
Map datacryo-ET of cryo-FIB milled yeast cell in which scs2/22 ist2 are deleted with high calcium levels; shown in Figures 5C and S3F
Sample
  • Cell: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are deleted, with high intracellular calcium; shown in Figures 5C and S3F
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsHoffmann PC / Bharat TAM / Wozny MR / Boulanger J / Miller EA / Kukulski W
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/8 United Kingdom
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MC_UP_1201/10 United Kingdom
CitationJournal: Dev Cell / Year: 2019
Title: Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures.
Authors: Patrick C Hoffmann / Tanmay A M Bharat / Michael R Wozny / Jerome Boulanger / Elizabeth A Miller / Wanda Kukulski /
Abstract: Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and ...Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways.
History
DepositionSep 11, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 13, 2019-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing site: PDBe / Status: Released

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Structure visualization

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

emd_10309.png

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Map

FileDownload / File: emd_10309.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS SIGNED BYTE
Annotationcryo-ET of cryo-FIB milled yeast cell in which scs2/22 ist2 are deleted with high calcium levels; shown in Figures 5C and S3F
Voxel sizeX=Y=Z: 7.4 Å
Density
Minimum - Maximum-128 - 127
Average (Standard dev.)6.4051456 (±16.537958)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-216
Dimensions18561920421
Spacing19201856421
CellA: 14208.0 Å / B: 13734.4 Å / C: 3115.4001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z7.47.47.4
M x/y/z19201856421
origin x/y/z0.0000.0000.000
length x/y/z14208.00013734.4003115.400
α/β/γ90.00090.00090.000
start NX/NY/NZ111-94150
NX/NY/NZ111123111
MAP C/R/S123
start NC/NR/NS00-216
NC/NR/NS19201856421
D min/max/mean-128.000127.0006.405

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Supplemental data

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Sample components

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Entire : cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are ...

EntireName: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are deleted, with high intracellular calcium; shown in Figures 5C and S3F
Components
  • Cell: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are deleted, with high intracellular calcium; shown in Figures 5C and S3F

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Supramolecule #1: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are ...

SupramoleculeName: cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are deleted, with high intracellular calcium; shown in Figures 5C and S3F
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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: Synthetic Complete -Trp medium with 15% high molecular weight dextran (w/v), 2 % glucose and 200 mM calcium chloride
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER
Details: 5 microliter of cell suspension was applied to the grid and then backside-blotted for 12-15 s.
DetailsCells were treated with 200 mM calcium chloride before plunge freezing. The cells with high GCaMP fluorescent signals, indicating high calcium levels, were targeted for cryo-FIB milling
Cryo protectantdextran
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 16 kV / Focused ion beam - Current: 0.023 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 103 K / Focused ion beam - Initial thickness: 5000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: Initial rough milling was performed at 30 kV 0.5-1 nA, then with 0.3 nA until 3000 nm thickness, then decreased to 0.1 nA to 1000 nm thickness. Fine milling to 150-300 nm ...Focused ion beam - Details: Initial rough milling was performed at 30 kV 0.5-1 nA, then with 0.3 nA until 3000 nm thickness, then decreased to 0.1 nA to 1000 nm thickness. Fine milling to 150-300 nm thickness was performed at 16kV 11 pA or 23 pA.. The value given for _emd_sectioning_focused_ion_beam.instrument is Scios DualBeam. 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
DetailsMontaged images of the grid were acquired at 200 nm pixel size to localize the lamellae on the grid. Overview montages of the individual lamellae were acquired at about 5 nm pixel size to assess the lamellae quality and identify ER-PM contact sites within the cells
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-4 / Average electron dose: 1.0 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. 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 micron defocus. A dose of 1.0 e-/square angstroms was applied per image of the tilt-series.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: eTomo / Details: 10 SIRT iterations / Number images used: 106
CTF correctionSoftware - Name: eTomo

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