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- EMDB-10710: Yeast Tomogram Collected on Lamella Generated by Fully Automated ... -

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Basic information

Entry
Database: EMDB / ID: EMD-10710
TitleYeast Tomogram Collected on Lamella Generated by Fully Automated FIB Milling
Map dataYeast Tomogram Collected on a Lamella Generated by Fully Automated FIB Milling
Sample
  • Cell: Yeast strain SK1
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsZachs T / Pilhofer M
Funding support Switzerland, European Union, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179255 Switzerland
European Research Council (ERC)679209European Union
CitationJournal: Elife / Year: 2020
Title: Fully automated, sequential focused ion beam milling for cryo-electron tomography.
Authors: Tobias Zachs / Andreas Schertel / João Medeiros / Gregor L Weiss / Jannik Hugener / Joao Matos / Martin Pilhofer /
Abstract: Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and ...Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
History
DepositionFeb 27, 2020-
Header (metadata) releaseMar 18, 2020-
Map releaseMar 18, 2020-
UpdateMar 18, 2020-
Current statusMar 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_10710.png

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Map

FileDownload / File: emd_10710.map.gz / Format: CCP4 / Size: 571.2 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationYeast Tomogram Collected on a Lamella Generated by Fully Automated FIB Milling
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
18.28 Å/pix.
x 231 pix.
= 4222.68 Å		18.28 Å/pix.
x 1266 pix.
= 23142.48 Å		18.28 Å/pix.
x 1024 pix.
= 18718.721 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: 18.28 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-579 - 300
Average (Standard dev.)1.6025327 (±12.318585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin209-208116
Dimensions12661024231
Spacing10241266231
CellA: 18718.72 Å / B: 23142.48 Å / C: 4222.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z18.28000097656318.2818.28
M x/y/z10241266231
origin x/y/z0.0000.0000.000
length x/y/z18718.72123142.4804222.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-208209116
NC/NR/NS10241266231
D min/max/mean-579.000300.0001.603

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Supplemental data

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Sample components

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Entire : Yeast strain SK1

EntireName: Yeast strain SK1
Components
  • Cell: Yeast strain SK1

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Supramolecule #1: Yeast strain SK1

SupramoleculeName: Yeast strain SK1 / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SK1

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE-PROPANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.005 nA / Focused ion beam - Duration: 1545 sec. / Focused ion beam - Temperature: 121 K / Focused ion beam - Initial thickness: 2000 nm / Focused ion beam - Final thickness: 243 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is Zeiss CrossBeam 550. This is not in a list of allowed values set(['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 BIOQUANTUM (6k x 4k) / Average electron dose: 1.96 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 61

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