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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 data
Samplecryo-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 (baker'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

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Map

FileDownload / File: emd_10309.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS SIGNED BYTE
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
Number of components: 1

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Component #1: cellular-component, cryo-ET of cryo-FIB milled yeast cell, in whi...

Cellular-componentName: 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
Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Cell / Method: cryo EM
Sample solutionBuffer solution: Synthetic Complete -Trp medium with 15% high molecular weight dextran (w/v), 2 % glucose and 200 mM calcium chloride
pH: 5.5
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K
Details: 5 microliter of cell suspension was applied to the grid and then backside-blotted for 12-15 s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Montaged 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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionDetails: 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.

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Image processing

ProcessingMethod: electron tomography / Number of sections: 106
3D reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software: eTomo / Details: 10 SIRT iterations

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