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- EMDB-4471: Cryo-SOFI enabling low-dose super-resolution correlative light an... -

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Entry
Database: EMDB / ID: EMD-4471
TitleCryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy
Map dataTomogram of pseudopod of an XC cell. This cell was imaged with cryo-fluorescence microscopy super-resolution optical fluctiation imaging (cryoSOFI) prior to tilt-series acquisition.
Sample
  • Cell: XC (rous sarcoma) cell transiently expressing mClover-TOM20
Biological speciesRattus (rat)
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_4471.png

Downloads & links

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Map

FileDownload / File: emd_4471.map.gz / Format: CCP4 / Size: 362.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationTomogram of pseudopod of an XC cell. This cell was imaged with cryo-fluorescence microscopy super-resolution optical fluctiation imaging (cryoSOFI) prior to tilt-series acquisition.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
16.88 Å/pix.
x 268 pix.
= 4523.84 Å		16.88 Å/pix.
x 849 pix.
= 14331.119 Å		16.88 Å/pix.
x 835 pix.
= 14094.799 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: 16.88 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-32767.0 - 32767.0
Average (Standard dev.)14363.378000000000611 (±1587.113499999999931)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-39-62110
Dimensions849835268
Spacing835849268
CellA: 14094.799 Å / B: 14331.119 Å / C: 4523.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z16.87999880239516.87999882214416.88
M x/y/z835849268
origin x/y/z0.0000.0000.000
length x/y/z14094.79914331.1194523.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-62-39110
NC/NR/NS835849268
D min/max/mean-32767.00032767.00014363.378

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Supplemental data

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Sample components

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Entire : XC (rous sarcoma) cell transiently expressing mClover-TOM20

EntireName: XC (rous sarcoma) cell transiently expressing mClover-TOM20
Components
  • Cell: XC (rous sarcoma) cell transiently expressing mClover-TOM20

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Supramolecule #1: XC (rous sarcoma) cell transiently expressing mClover-TOM20

SupramoleculeName: XC (rous sarcoma) cell transiently expressing mClover-TOM20
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Rattus (rat) / 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
GridMaterial: GOLD / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: LACEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER
DetailsXC (rous sarcoma) cell 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: 31
CTF correctionSoftware - Name: CTFPHASEFLIP

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