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- EMDB-4756: Subtomogram average of cytosolic ribosomes after cryo-FIB lift-ou... -

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Basic information

Entry
Database: EMDB / ID: EMD-4756
TitleSubtomogram average of cytosolic ribosomes after cryo-FIB lift-out (p1-pre state)
Map data
SampleCytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-pre state)
Biological speciesCaenorhabditis elegans (roundworm)
Methodsubtomogram averaging / cryo EM / Resolution: 12.3 Å
AuthorsPfeffer S / Mahamid J / Albert S / Plitzko JM
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research FoundationExcellence Clusters CIPSM Germany
German Research FoundationSFB 1035 Germany
CitationJournal: Nat. Methods / Year: 2019
Title: A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.
Authors: Miroslava Schaffer / Stefan Pfeffer / Julia Mahamid / Stephan Kleindiek / Tim Laugks / Sahradha Albert / Benjamin D Engel / Andreas Rummel / Andrew J Smith / Wolfgang Baumeister / Juergen M Plitzko /
Abstract: Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows ...Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.
History
DepositionApr 1, 2019-
Header (metadata) releaseJul 10, 2019-
Map releaseJul 10, 2019-
UpdateJan 22, 2020-
Current statusJan 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4756.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-0.47256115 - 1.2170705
Average (Standard dev.)0.011816717 (±0.16820337)
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-0.4731.2170.012

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Supplemental data

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Sample components

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Entire Cytosolic ribosome in the native cellular environment after cryo-...

EntireName: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-pre state)
Number of components: 1

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Component #1: protein, Cytosolic ribosome in the native cellular environment af...

ProteinName: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-pre state)
Recombinant expression: No
SourceSpecies: Caenorhabditis elegans (roundworm)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Tissue / Method: cryo EM
Sample solutionpH: 7.3
VitrificationCryogen name: NITROGEN
Details: The sample was high pressure frozen in a BAL-TEC HPM 100 high-pressure freezer..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 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 processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 2862
3D reconstructionSoftware: RELION / Resolution: 12.3 Å / Resolution method: FSC 0.143 CUT-OFF

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