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- EMDB-10310: cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overe... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-10310 | ||||||||||||
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Title | cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overexpressed and scs2/22 ist2 tcb1/2 are deleted; shown in Figure 6 of publication | ||||||||||||
![]() | cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 tcb1/2 are deleted and Tcb3-GFP is overexpressed; shown in Figures 6 of publication | ||||||||||||
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Biological species | ![]() ![]() | ||||||||||||
Method | electron tomography / cryo EM | ||||||||||||
![]() | Hoffmann PC / Bharat TAM / Wozny MR / Boulanger J / Miller EA / Kukulski W | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1.2 GB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.2 KB 13.2 KB | Display Display | ![]() |
Images | ![]() | 143.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article ( |
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EM raw data | ![]() Data size: 3.1 Data #1: cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overexpressed and scs2/22 ist2 tcb1/2 are deleted [tilt series]) |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | cryo-ET of cryo-FIB milled yeast cell, in which scs2/22 ist2 tcb1/2 are deleted and Tcb3-GFP is overexpressed; shown in Figures 6 of publication | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 7.4 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overe...
Entire | Name: cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overexpressed and scs2/22 ist2 tcb1/2 are deleted; shown in Figures 6 of publication |
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Components |
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-Supramolecule #1: cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overe...
Supramolecule | Name: cryo-ET of cryo-FIB milled yeast cell, in which Tcb3-GFP is overexpressed and scs2/22 ist2 tcb1/2 are deleted; shown in Figures 6 of publication type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | electron tomography |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 5.5 Details: Synthetic Complete -Trp medium with 15% high molecular weight dextran (w/v), 1 % raffinose and 1% galactose |
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Vitrification | Cryogen 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. |
Details | Cells were induced by addition of 1% galactose for 24h before plunge-freezing |
Cryo protectant | dextran |
Sectioning | Focused 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: 230 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
Microscope | FEI TITAN KRIOS |
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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 |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.2 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 -4.5 micron defocus. A dose of 1.2 e-/square angstroms was applied per image of the tilt-series. |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: eTomo / Details: 10 SIRT iterations / Number images used: 117 |
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CTF correction | Software - Name: eTomo |