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- EMDB-21039: Cryo-electron tomogram of FIB-milled U2OS cells -

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Basic information

Entry
Database: EMDB / ID: EMD-21039
TitleCryo-electron tomogram of FIB-milled U2OS cells
Map dataRepresentative cryo-FIB milled mammalian cell tomogram
Sample
  • Cell: FIB-milled frozen human osteosarcoma U2OS cell
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM / Resolution: 12.28 Å
AuthorsWatanabe R / Villa E
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/Office of the Director1DP2GM123494-01 United States
National Science Foundation (NSF, United States)ECCS-1542148 United States
CitationJournal: Nat Protoc / Year: 2020
Title: Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography.
Authors: Felix R Wagner / Reika Watanabe / Ruud Schampers / Digvijay Singh / Hans Persoon / Miroslava Schaffer / Peter Fruhstorfer / Jürgen Plitzko / Elizabeth Villa /
Abstract: Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron ...Recent advances have made cryogenic (cryo) electron microscopy a key technique to achieve near-atomic-resolution structures of biochemically isolated macromolecular complexes. Cryo-electron tomography (cryo-ET) can give unprecedented insight into these complexes in the context of their natural environment. However, the application of cryo-ET is limited to samples that are thinner than most cells, thereby considerably reducing its applicability. Cryo-focused-ion-beam (cryo-FIB) milling has been used to carve (micromachining) out 100-250-nm-thin regions (called lamella) in the intact frozen cells. This procedure opens a window into the cells for high-resolution cryo-ET and structure determination of biomolecules in their native environment. Further combination with fluorescence microscopy allows users to determine cells or regions of interest for the targeted fabrication of lamellae and cryo-ET imaging. Here, we describe how to prepare lamellae using a microscope equipped with both FIB and scanning electron microscopy modalities. Such microscopes (Aquilos Cryo-FIB/Scios/Helios or CrossBeam) are routinely referred to as dual-beam microscopes, and they are equipped with a cryo-stage for all operations in cryogenic conditions. The basic principle of the described methodologies is also applicable for other types of dual-beam microscopes equipped with a cryo-stage. We also briefly describe how to integrate fluorescence microscopy data for targeted milling and critical considerations for cryo-ET data acquisition of the lamellae. Users familiar with cryo-electron microscopy who get basic training in dual-beam microscopy can complete the protocol within 2-3 d, allowing for several pause points during the procedure.
History
DepositionNov 25, 2019-
Header (metadata) releaseDec 25, 2019-
Map releaseMay 27, 2020-
UpdateJun 17, 2020-
Current statusJun 17, 2020Processing site: RCSB / 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_21039.png

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Map

FileDownload / File: emd_21039.map.gz / Format: CCP4 / Size: 365.1 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationRepresentative cryo-FIB milled mammalian cell tomogram
Voxel sizeX=Y=Z: 12.29 Å
Density
Minimum - Maximum-127 - 127
Average (Standard dev.)-19.314962 (±108.33787)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin12100
Dimensions12801236242
Spacing12361280242
CellA: 15190.44 Å / B: 15731.2 Å / C: 2974.18 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z12.2912.2912.29
M x/y/z12361280242
origin x/y/z0.0000.0000.000
length x/y/z15190.44015731.2002974.180
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS01210
NC/NR/NS12361280242
D min/max/mean-127.000127.000-19.315

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Supplemental data

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Sample components

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Entire : FIB-milled frozen human osteosarcoma U2OS cell

EntireName: FIB-milled frozen human osteosarcoma U2OS cell
Components
  • Cell: FIB-milled frozen human osteosarcoma U2OS cell

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Supramolecule #1: FIB-milled frozen human osteosarcoma U2OS cell

SupramoleculeName: FIB-milled frozen human osteosarcoma U2OS cell / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Organ: bone / Tissue: epithelial cell

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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
GridModel: Quantifoil R2/1 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.03 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 90 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is TFS Scios. 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 POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.28 Å / Software - Name: eTomo / Number images used: 66

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