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- EMDB-4870: In situ cryo-electron tomogram after cryo-FIB lift-out (from lame... -

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Basic information

Entry
Database: EMDB / ID: EMD-4870
TitleIn situ cryo-electron tomogram after cryo-FIB lift-out (from lamella 2)
Map dataIn situ cryo-electron tomogram after cryo-FIB lift-out (from lamella 2)
Sample
  • Tissue: Native cellular environment after cryo-FIB lift-out from C. elegans (from lamella 2)
Biological speciesCaenorhabditis elegans (invertebrata)
Methodelectron tomography / cryo EM
AuthorsSchaffer M / Pfeffer 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 15, 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
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_4870.png

Downloads & links

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Map

FileDownload / File: emd_4870.map.gz / Format: CCP4 / Size: 762.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationIn situ cryo-electron tomogram after cryo-FIB lift-out (from lamella 2)
Voxel sizeX=Y=Z: 13.68 Å
Density
Minimum - Maximum-267 - 159
Average (Standard dev.)1.4884288 (±15.308075)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-232
Dimensions928928464
Spacing928928464
CellA: 12695.04 Å / B: 12695.04 Å / C: 6347.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.6813.6813.68
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z12695.04012695.0406347.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-232
NC/NR/NS928928464
D min/max/mean-267.000159.0001.488

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Supplemental data

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Sample components

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Entire : Native cellular environment after cryo-FIB lift-out from C. elega...

EntireName: Native cellular environment after cryo-FIB lift-out from C. elegans (from lamella 2)
Components
  • Tissue: Native cellular environment after cryo-FIB lift-out from C. elegans (from lamella 2)

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Supramolecule #1: Native cellular environment after cryo-FIB lift-out from C. elega...

SupramoleculeName: Native cellular environment after cryo-FIB lift-out from C. elegans (from lamella 2)
type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.3
VitrificationCryogen name: NITROGEN
DetailsAdult C. elegans worms.
High pressure freezingInstrument: OTHER
Details: The value given for _emd_high_pressure_freezing.instrument is BAL-TEC HPM 100 (Leica). This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID ...Details: The value given for _emd_high_pressure_freezing.instrument is BAL-TEC HPM 100 (Leica). This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.05 nA / Focused ion beam - Duration: 3500 sec. / Focused ion beam - Temperature: 92 K / Focused ion beam - Initial thickness: 3000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: FIB instrument equipped with a Quorum PP3000T cryo-system (Quorum Technologies) and a Kleindiek MM3A-EM micromanipulator (Kleindiek Nanotechnik GmbH).. The value given for ...Focused ion beam - Details: FIB instrument equipped with a Quorum PP3000T cryo-system (Quorum Technologies) and a Kleindiek MM3A-EM micromanipulator (Kleindiek Nanotechnik GmbH).. The value given for _emd_sectioning_focused_ion_beam.instrument is FIB Quanta 3D FEG (Thermo Fisher). 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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm 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

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 39

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