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- EMDB-4372: Ribosome subtomogram average obtained for control yeast cells -

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Basic information

Entry
Database: EMDB / ID: EMD-4372
TitleRibosome subtomogram average obtained for control yeast cells
Map dataRibosome subtomogram average obtained for control yeast cells
Sample
  • Complex: Cytosolic 80S ribosome
Biological speciesSaccharomyces cerevisiae W303 (yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 11.5 Å
AuthorsPfeffer S / Engel BD / Schaffer M
CitationJournal: 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
DepositionApr 16, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseJun 27, 2018-
UpdateJul 25, 2018-
Current statusJul 25, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4372.png

Downloads & links

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Map

FileDownload / File: emd_4372.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome subtomogram average obtained for control yeast cells
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.42 Å/pix.
x 128 pix.
= 437.76 Å		3.42 Å/pix.
x 128 pix.
= 437.76 Å		3.42 Å/pix.
x 128 pix.
= 437.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.42 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-1.098915 - 1.5347656
Average (Standard dev.)0.0008373331 (±0.14218503)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 437.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z437.760437.760437.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-1.0991.5350.001

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Supplemental data

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Sample components

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Entire : Cytosolic 80S ribosome

EntireName: Cytosolic 80S ribosome
Components
  • Complex: Cytosolic 80S ribosome

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Supramolecule #1: Cytosolic 80S ribosome

SupramoleculeName: Cytosolic 80S ribosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV / Details: 10 seconds blot time, blot force 10.
DetailsCytosolic 80S ribosomes imaged in control yeast cells thinned by focused ion beam milling

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 6.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 36974
ExtractionNumber tomograms: 14 / Number images used: 36974 / Method: template matching + cross-correlation cutoff / Software - Name: PyTom
CTF correctionSoftware - Name: PyTom
Final angle assignmentType: OTHER

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