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- EMDB-17245: C. elegans L1 larva 80S ribosome class 4 -

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Basic information

Entry
Database: EMDB / ID: EMD-17245
TitleC. elegans L1 larva 80S ribosome class 4
Map dataC. elegans L1 larva ribosome class 4
Sample
  • Complex: C. elegans L1 larva, CGC strain NK2476
KeywordsCaenorhabditis / elegans / ribosome / 80S / L1 / larva / C. elegans
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsubtomogram averaging / cryo EM / Resolution: 8.4 Å
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: 2023
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-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17245.png

Downloads & links

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Map

FileDownload / File: emd_17245.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC. elegans L1 larva ribosome class 4
Voxel sizeX=Y=Z: 2.98 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.22722611 - 1.1387715
Average (Standard dev.)0.022490218 (±0.12202458)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 476.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17245_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Additional map: postprocessed map

Fileemd_17245_additional_1.map
Annotationpostprocessed map
Projections & Slices
AxesZYX

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Histogram

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Half map: Half map 1

Fileemd_17245_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_17245_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : C. elegans L1 larva, CGC strain NK2476

EntireName: C. elegans L1 larva, CGC strain NK2476
Components
  • Complex: C. elegans L1 larva, CGC strain NK2476

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Supramolecule #1: C. elegans L1 larva, CGC strain NK2476

SupramoleculeName: C. elegans L1 larva, CGC strain NK2476 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / Strain: NK2476 / Tissue: Whole L1 larva / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statetissue

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Sample preparation

BufferpH: 7.5 / Details: M9 buffer + 20% Ficoll 400
GridModel: Homemade / 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1 / Average exposure time: 0.68 sec. / Average electron dose: 3.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 200 / Number images used: 65451
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number subtomograms used: 20638
FSC plot (resolution estimation)

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