|Entry||Database: EMDB / ID: EMD-10309|
|Title||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|
|Sample||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 species||Saccharomyces cerevisiae (baker's yeast)|
|Method||electron tomography / cryo EM|
|Authors||Hoffmann PC / Bharat TAM / Wozny MR / Boulanger J / Miller EA / Kukulski W|
|Funding support|| United Kingdom, 3 items |
|Citation||Journal: 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.
Downloads & links
|File||Download / File: emd_10309.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS SIGNED BYTE|
|Voxel size||X=Y=Z: 7.4 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 are ...
|Entire||Name: 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
-Component #1: cellular-component, cryo-ET of cryo-FIB milled yeast cell, in whi...
|Cellular-component||Name: 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
|Source||Species: Saccharomyces cerevisiae (baker's yeast)|
|Specimen||Specimen state: Cell / Method: cryo EM|
|Sample solution||Buffer solution: Synthetic Complete -Trp medium with 15% high molecular weight dextran (w/v), 2 % glucose and 200 mM calcium chloride|
|Vitrification||Instrument: 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.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: 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 gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||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.|
|Processing||Method: electron tomography / Number of sections: 106|
|3D reconstruction||Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software: eTomo / Details: 10 SIRT iterations|
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