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- EMDB-4472: Cryo-SOFI enabling low-dose super-resolution correlative light an... -

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Basic information

Entry
Database: EMDB / ID: EMD-4472
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 (rous sarcoma) cell transiently expressing Lifeact-Dendra2
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
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_4472.png

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Map

FileDownload / File: emd_4472.map.gz / Format: CCP4 / Size: 708.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLow-magnification tomogram for correlation with cryoSOFI data.
Voxel sizeX=Y=Z: 46.2 Å
Density
Minimum - Maximum-14832.860000000000582 - 6635.067399999999907
Average (Standard dev.)16.667002 (±240.935869999999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-173-16943
Dimensions1515149582
Spacing1495151582
CellA: 69069.0 Å / B: 69993.0 Å / C: 3788.4001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z46.246.246.2
M x/y/z1495151582
origin x/y/z0.0000.0000.000
length x/y/z69069.00069993.0003788.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-169-17343
NC/NR/NS1495151582
D min/max/mean-14832.8606635.06716.667

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Supplemental data

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Sample components

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

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

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

SupramoleculeName: XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2
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
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
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 FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 50000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
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
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: 40

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