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- EMDB-33910: In situ cryo-electron tomogram of collagen fibril in fibrosis liv... -

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Basic information

Entry
Database: EMDB / ID: EMD-33910
TitleIn situ cryo-electron tomogram of collagen fibril in fibrosis liver tissue 1
Map data
Sample
  • Tissue: collagen fibril in fibrosis liver tissue
Keywordscollagen fibril / fibrosis / liver tissue / CryoET / PROTEIN FIBRIL
Biological speciesMus musculus (house mouse)
Methodelectron tomography
AuthorsWang S / Zhou H / Chen W / Jiang Y / Yan X / You H / Li X
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Rep / Year: 2023
Title: CryoFIB milling large tissue samples for cryo-electron tomography.
Authors: Sihan Wang / Heng Zhou / Wei Chen / Yifeng Jiang / Xuzhen Yan / Hong You / Xueming Li /
Abstract: Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In ...Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow-ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.
History
DepositionJul 26, 2022-
Header (metadata) releaseNov 9, 2022-
Map releaseNov 9, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33910.png

Downloads & links

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Map

FileDownload / File: emd_33910.map.gz / Format: CCP4 / Size: 576.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
12.72 Å/pix.
x 231 pix.
= 2938.32 Å		12.72 Å/pix.
x 960 pix.
= 12211.2 Å		12.72 Å/pix.
x 682 pix.
= 8675.04 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: 12.72 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-38.720832999999999 - 20.164597000000001
Average (Standard dev.)0.53733504 (±1.5116415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin114-138138
Dimensions960682231
Spacing682960231
CellA: 8675.04 Å / B: 12211.2 Å / C: 2938.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : collagen fibril in fibrosis liver tissue

EntireName: collagen fibril in fibrosis liver tissue
Components
  • Tissue: collagen fibril in fibrosis liver tissue

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Supramolecule #1: collagen fibril in fibrosis liver tissue

SupramoleculeName: collagen fibril in fibrosis liver tissue / type: tissue / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
GridModel: Homemade / Material: COPPER / Mesh: 150 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
High pressure freezingInstrument: OTHER
Details: The value given for _em_high_pressure_freezing.instrument is Leica HPM100. This is not in a list of allowed values {'LEICA EM HPM100', 'BAL-TEC HPM 010', 'LEICA EM PACT2', 'EMS-002 RAPID ...Details: The value given for _em_high_pressure_freezing.instrument is Leica HPM100. This is not in a list of allowed values {'LEICA EM HPM100', 'BAL-TEC HPM 010', 'LEICA EM PACT2', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM PACT'} so OTHER is written into the XML file.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.04 / Focused ion beam - Duration: 21600 / Focused ion beam - Temperature: 103 K / Focused ion beam - Initial thickness: 30000 / Focused ion beam - Final thickness: 130
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Helios Nanolab G3 UC. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm
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 reconstructionNumber images used: 58

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