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- EMDB-18187: Focused refinment of 11-protofilament microtubule from C. elegans -

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Basic information

Entry
Database: EMDB / ID: EMD-18187
TitleFocused refinment of 11-protofilament microtubule from C. elegans
Map dataPostprocessed density masked
Sample
  • Tissue: C. elegans L1 larva, CGC strain NK2476
KeywordsCaenorhabditis / elegans / touch receptor cell / microtubule / L1 / larva / C. elegans / STRUCTURAL PROTEIN
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsubtomogram averaging / cryo EM / Resolution: 13.0 Å
AuthorsSchioetz OH / Kaiser CJO / Klumpe S / Klebl DP / Schneider J / 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
DepositionAug 11, 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_18187.png

Downloads & links

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Map

FileDownload / File: emd_18187.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed density masked
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
3.78 Å/pix.
x 64 pix.
= 241.92 Å		3.78 Å/pix.
x 64 pix.
= 241.92 Å		3.78 Å/pix.
x 64 pix.
= 241.92 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.78 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.277347 - 0.50585276
Average (Standard dev.)0.016305096 (±0.09144963)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 241.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18187_msk_1.map
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Additional map: postprocessed density unmasked

Fileemd_18187_additional_1.map
Annotationpostprocessed density unmasked
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Additional map: Raw density without postprocessing

Fileemd_18187_additional_2.map
AnnotationRaw density without postprocessing
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Half map: Half map 2

Fileemd_18187_half_map_1.map
AnnotationHalf map 2
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Half map: Half set 1

Fileemd_18187_half_map_2.map
AnnotationHalf set 1
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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
  • Tissue: 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: tissue / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / Strain: NK2476 / Tissue: Whole L1 larva

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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
VitrificationCryogen name: NITROGEN / Details: High pressure freezing, Leica EM ICE.
DetailsDevelopmentally arrested L1 larvae

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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 real images: 41 / Average exposure time: 0.73 sec. / Average electron dose: 3.23 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: 64000
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 20516
ExtractionNumber tomograms: 12 / Number images used: 20220
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software: (Name: STOPGAP (ver. 0.7), RELION (ver. 3.0))
FSC plot (resolution estimation)

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