|Entry||Database: EMDB / ID: EMD-4755|
|Title||Subtomogram average of cytosolic ribosomes after cryo-FIB lift-out (p1-post state)|
|Sample||Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-post state)|
|Biological species||Caenorhabditis elegans (roundworm)|
|Method||subtomogram averaging / cryo EM / Resolution: 13.7 Å|
|Authors||Pfeffer S / Mahamid J / Albert S / Plitzko JM|
|Funding support|| Germany, 2 items |
|Citation||Journal: 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.
|Structure viewer||EM map: |
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|File||Download / File: emd_4755.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 3.42 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Cytosolic ribosome in the native cellular environment after cryo-...
|Entire||Name: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-post state)|
Number of components: 1
-Component #1: protein, Cytosolic ribosome in the native cellular environment af...
|Protein||Name: Cytosolic ribosome in the native cellular environment after cryo-FIB lift-out from C. elegans (p1-post state)|
Recombinant expression: No
|Source||Species: Caenorhabditis elegans (roundworm)|
|Specimen||Specimen state: Tissue / Method: cryo EM|
|Sample solution||pH: 7.3|
|Vitrification||Cryogen name: NITROGEN|
Details: The sample was high pressure frozen in a BAL-TEC HPM 100 high-pressure freezer..
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 2033|
|3D reconstruction||Software: RELION / Resolution: 13.7 Å / Resolution method: FSC 0.143 CUT-OFF|
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