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- EMDB-50608: SOLIST cryo-tomogram of human brain organoid #2 -

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Basic information

Entry
Database: EMDB / ID: EMD-50608
TitleSOLIST cryo-tomogram of human brain organoid #2
Map data
Sample
  • Cell: cryo-tomogram of human brain organoid #2
Keywordsbrain / organoid / microtubule / ribosome / mitochondria / UNKNOWN FUNCTION
Biological speciesHomo sapiens (human)
Methodelectron tomography
AuthorsErdmann PS / Nguyen HTD / Perone G / Klena N / Vazzana R / Kaluthantrige Don F / Silva M / Sorrentino S / Swuec P / Leroux F ...Erdmann PS / Nguyen HTD / Perone G / Klena N / Vazzana R / Kaluthantrige Don F / Silva M / Sorrentino S / Swuec P / Leroux F / Kalebic N / Coscia F
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Methods / Year: 2024
Title: Serialized on-grid lift-in sectioning for tomography (SOLIST) enables a biopsy at the nanoscale.
Authors: Ho Thuy Dung Nguyen / Gaia Perone / Nikolai Klena / Roberta Vazzana / Flaminia Kaluthantrige Don / Malan Silva / Simona Sorrentino / Paolo Swuec / Frederic Leroux / Nereo Kalebic / Francesca ...Authors: Ho Thuy Dung Nguyen / Gaia Perone / Nikolai Klena / Roberta Vazzana / Flaminia Kaluthantrige Don / Malan Silva / Simona Sorrentino / Paolo Swuec / Frederic Leroux / Nereo Kalebic / Francesca Coscia / Philipp S Erdmann /
Abstract: Cryo-focused ion beam milling has substantially advanced our understanding of molecular processes by opening windows into cells. However, applying this technique to complex samples, such as tissues, ...Cryo-focused ion beam milling has substantially advanced our understanding of molecular processes by opening windows into cells. However, applying this technique to complex samples, such as tissues, has presented considerable technical challenges. Here we introduce an innovative adaptation of the cryo-lift-out technique, serialized on-grid lift-in sectioning for tomography (SOLIST), addressing these limitations. SOLIST enhances throughput, minimizes ice contamination and improves sample stability for cryo-electron tomography. It thereby facilitates the high-resolution imaging of a wide range of specimens. We illustrate these advantages on reconstituted liquid-liquid phase-separated droplets, brain organoids and native tissues from the mouse brain, liver and heart. With SOLIST, cellular processes can now be investigated at molecular resolution directly in native tissue. Furthermore, our method has a throughput high enough to render cryo-lift-out a competitive tool for structural biology. This opens new avenues for unprecedented insights into cellular function and structure in health and disease, a 'biopsy at the nanoscale'.
History
DepositionJun 10, 2024-
Header (metadata) releaseSep 11, 2024-
Map releaseSep 11, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50608.png

Downloads & links

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Map

FileDownload / File: emd_50608.map.gz / Format: CCP4 / Size: 256 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
15.62 Å/pix.
x 256 pix.
= 3999.744 Å		15.62 Å/pix.
x 512 pix.
= 7999.488 Å		15.62 Å/pix.
x 512 pix.
= 7999.488 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: 15.624 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-26.720852000000001 - 12.148104
Average (Standard dev.)0.27360097 (±0.50748783)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-128
Dimensions512512256
Spacing512512256
CellA: 7999.488 Å / B: 7999.488 Å / C: 3999.744 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : cryo-tomogram of human brain organoid #2

EntireName: cryo-tomogram of human brain organoid #2
Components
  • Cell: cryo-tomogram of human brain organoid #2

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Supramolecule #1: cryo-tomogram of human brain organoid #2

SupramoleculeName: cryo-tomogram of human brain organoid #2 / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: H9 ESC / Organ: brain / Tissue: corpus callosum

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.5
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
Details: glow-discharged at 30 mA for 30 s (GloQube Plus Glow discharge system, Quorum Technologies)
DetailsSOLIST cryo-tomogram of human brain organoid #2
High pressure freezingInstrument: OTHER
Details: Before use, 3 mm type A (100 micrometer well) and type B planchettes were cleaned by sonication for 5 min, 30 s on, 30 s off, 40% Amplitude on a Branson SFX 550. Type B lids were polished ...Details: Before use, 3 mm type A (100 micrometer well) and type B planchettes were cleaned by sonication for 5 min, 30 s on, 30 s off, 40% Amplitude on a Branson SFX 550. Type B lids were polished with 1 micrometer lapping paper and coated with 0.1% soy lecithin dissolved in chloroform.. 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% dextran, 5% sucrose
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: 60 / Focused ion beam - Temperature: 80 K / Focused ion beam - Initial thickness: 4000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: SOLIST procedure. The value given for _em_focused_ion_beam.instrument is 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

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 41 / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 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 reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo (ver. 4.11.7) / Details: denoised with CryoCare 2.1 / Number images used: 41

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