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- EMDB-17247: C. elegans L1 larva ventral pharyngeal periphery tomogram -

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Basic information

Entry
Database: EMDB / ID: EMD-17247
TitleC. elegans L1 larva ventral pharyngeal periphery tomogram
Map dataBin4 pharyngeal marginal cell tomogram
Sample
  • Tissue: C. elegans L1 larva, CGC strain AM140
KeywordsCaenorhabditis / elegans / L1 / larva / C. elegans / pharynx / marginal cell / neuron cell bodies / pharyngeal mucle / basal lamina / CYTOSOLIC PROTEIN
Biological speciesCaenorhabditis elegans (invertebrata)
Methodelectron tomography / cryo EM
AuthorsSchioetz OH / Kaiser CJO / Klumpe S / Beck F / Plitzko JM
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Methods / Year: 2024
Title: Serial Lift-Out: sampling the molecular anatomy of whole organisms.
Authors: Oda Helene Schiøtz / Christoph J O Kaiser / Sven Klumpe / Dustin R Morado / Matthias Poege / Jonathan Schneider / Florian Beck / David P Klebl / Christopher Thompson / Jürgen M Plitzko /
Abstract: Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. ...Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
History
DepositionApr 28, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17247.png

Downloads & links

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Map

FileDownload / File: emd_17247.map.gz / Format: CCP4 / Size: 2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBin4 pharyngeal marginal cell tomogram
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
11.72 Å/pix.
x 512 pix.
= 6000.64 Å		11.72 Å/pix.
x 1024 pix.
= 12001.28 Å		11.72 Å/pix.
x 1024 pix.
= 12001.28 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 11.72 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3221110.0 - 4178742.799999999813735
Average (Standard dev.)17765.869999999998981 (±454921.96999999997206)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-44
Dimensions10241024512
Spacing10241024512
CellA: 12001.28 Å / B: 12001.28 Å / C: 6000.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : C. elegans L1 larva, CGC strain AM140

EntireName: C. elegans L1 larva, CGC strain AM140
Components
  • Tissue: C. elegans L1 larva, CGC strain AM140

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Supramolecule #1: C. elegans L1 larva, CGC strain AM140

SupramoleculeName: C. elegans L1 larva, CGC strain AM140 / type: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / Strain: AM140 / Tissue: Whole L1 larva

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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: 7.5 / Details: M9 buffer + 20% Ficoll 400
GridModel: Homemade / Material: COPPER / Mesh: 50 / Support film - Material: FORMVAR / Support film - topology: CONTINUOUS / Support film - Film thickness: 100
VitrificationCryogen name: NITROGEN / Details: High pressure freezing, Leica EM ICE.
DetailsDevelopmentally arrested L1 larvae
High pressure freezingInstrument: OTHER
Details: The value given for _em_high_pressure_freezing.instrument is Leica EM ICE. This is not in a list of allowed values {'LEICA EM HPM100', 'LEICA EM PACT', 'LEICA EM PACT2', 'BAL-TEC HPM 010', ...Details: The value given for _em_high_pressure_freezing.instrument is Leica EM ICE. This is not in a list of allowed values {'LEICA EM HPM100', 'LEICA EM PACT', 'LEICA EM PACT2', 'BAL-TEC HPM 010', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER'} so OTHER is written into the XML file.
Cryo protectant20% Ficoll 400
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 1800 / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 250
Focused ion beam - Details: Serial Lift-Out of 160,000 nm original volume sectioned into 4000 nm slices, thinned to roughly 250 nm.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos ...Focused ion beam - Details: Serial Lift-Out of 160,000 nm original volume sectioned into 4000 nm slices, thinned to roughly 250 nm.. The value given for _em_focused_ion_beam.instrument is TFS Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 48 / Average exposure time: 2.24 sec. / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 42000
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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.12.32) / Details: ARETOMO 1.3.3 tilt series alignment / Number images used: 48

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