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Yorodumi- EMDB-4375: GEM subtomogram average obtained for yeast cells treated with rap... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4375 | |||||||||
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Title | GEM subtomogram average obtained for yeast cells treated with rapamycin | |||||||||
Map data | GEM subtomogram average obtained for yeast cells treated with rapamycin | |||||||||
Sample |
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Biological species | Saccharomyces cerevisiae W303 (yeast) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 26.3 Å | |||||||||
Authors | Pfeffer S / Engel BD / Schaffer M | |||||||||
Citation | Journal: Cell / Year: 2018 Title: mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding. Authors: M Delarue / G P Brittingham / S Pfeffer / I V Surovtsev / S Pinglay / K J Kennedy / M Schaffer / J I Gutierrez / D Sang / G Poterewicz / J K Chung / J M Plitzko / J T Groves / C Jacobs- ...Authors: M Delarue / G P Brittingham / S Pfeffer / I V Surovtsev / S Pinglay / K J Kennedy / M Schaffer / J I Gutierrez / D Sang / G Poterewicz / J K Chung / J M Plitzko / J T Groves / C Jacobs-Wagner / B D Engel / L J Holt / Abstract: Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed ...Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4375.map.gz | 28.7 MB | EMDB map data format | |
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Header (meta data) | emd-4375-v30.xml emd-4375.xml | 11.5 KB 11.5 KB | Display Display | EMDB header |
Images | emd_4375.png | 81.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4375 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4375 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_4375.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | GEM subtomogram average obtained for yeast cells treated with rapamycin | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.42 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Genetically Encoded Multimeric nanoparticles (GEMs)
Entire | Name: Genetically Encoded Multimeric nanoparticles (GEMs) |
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Components |
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-Supramolecule #1: Genetically Encoded Multimeric nanoparticles (GEMs)
Supramolecule | Name: Genetically Encoded Multimeric nanoparticles (GEMs) / type: complex / ID: 1 / Parent: 0 Details: GEMs are homomultimeric scaffolds (Pyrococcus furious encapsulin) fused to a fluorescent protein (T- Sapphire fluorophore). GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. |
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Source (natural) | Organism: Saccharomyces cerevisiae W303 (yeast) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7 |
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: 10 seconds blot time, blot force 10. |
Details | GEMs imaged in rapamycin-treated yeast cells thinned by focused ion beam milling |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Extraction | Number tomograms: 13 / Number images used: 74 / Method: template matching + visual inspection / Software - Name: PyTom |
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CTF correction | Software - Name: PyTom |
Final angle assignment | Type: OTHER |
Final reconstruction | Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 26.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 74 |