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- EMDB-4473: Cryo-SOFI enabling low-dose super-resolution correlative light an... -

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Basic information

Entry
Database: EMDB / ID: EMD-4473
TitleCryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy
Map dataLow-magnification tomogram for correlation with cryoSOFI data.
Sample
  • Cell: XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2
Biological speciesRattus (rats)
Methodelectron tomography / cryo EM
AuthorsMoser F / Prazak V / Mordhorst V / Andrade AM / Baker LA / Hagen C / Grunewald K / Kaufmann R
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.
Authors: Felipe Moser / Vojtěch Pražák / Valerie Mordhorst / Débora M Andrade / Lindsay A Baker / Christoph Hagen / Kay Grünewald / Rainer Kaufmann /
Abstract: Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context ...Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.
History
DepositionDec 16, 2018-
Header (metadata) releaseFeb 27, 2019-
Map releaseFeb 27, 2019-
UpdateMar 20, 2019-
Current statusMar 20, 2019Processing site: PDBe / Status: Released

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Structure visualization

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

emd_4473.png

Downloads & links

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Map

FileDownload / File: emd_4473.map.gz / Format: CCP4 / Size: 839.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationLow-magnification tomogram for correlation with cryoSOFI data.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
46.2 Å/pix.
x 156 pix.
= 7207.2 Å		46.2 Å/pix.
x 1761 pix.
= 81358.203 Å		46.2 Å/pix.
x 1602 pix.
= 74012.398 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: 46.2 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-32768.0 - 32766.0
Average (Standard dev.)11238.443999999999505 (±628.056699999999978)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-46-27852
Dimensions17611602156
Spacing16021761156
CellA: 74012.4 Å / B: 81358.2 Å / C: 7207.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z46.19999875156146.20000170357846.2
M x/y/z16021761156
origin x/y/z0.0000.0000.000
length x/y/z74012.39881358.2037207.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-278-4652
NC/NR/NS16021761156
D min/max/mean-32768.00032766.00011238.444

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Supplemental data

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Sample components

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Entire : XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2

EntireName: XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2
Components
  • Cell: XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2

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Supramolecule #1: XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2

SupramoleculeName: XC cell(rous sarcoma) transiently expressing Lifeact-Dendra2
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Rattus (rats) / Tissue: muscle

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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: 7.4 / Details: DMEM 10% fetal bofine serum
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
DetailsXC (rous sarcoma) transiently expressing mClover-TOM20
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3836 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-20 / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 6.03 µm / Calibrated defocus min: 2.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 50000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo / Number images used: 39

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