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

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Database: EMDB / ID: 4472
TitleCryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy
Map dataLow-magnification tomogram for correlation with cryoSOFI data.
SampleXC (rous sarcoma) cell transiently expressing Lifeact-Dendra2:
SourceRattus (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.
DateDeposition: Dec 16, 2018 / Header (metadata) release: Feb 27, 2019 / Map release: Feb 27, 2019 / Last update: Mar 20, 2019

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Structure visualization

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

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Map

Fileemd_4472.map.gz (map file in CCP4 format, 742896 KB)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
82 pix
46.2 Å/pix.
= 3788.4 Å
1515 pix
46.2 Å/pix.
= 69993. Å
1495 pix
46.2 Å/pix.
= 69069. Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 46.2 Å
Density
Minimum - Maximum-14832.860000000000582 - 6635.067399999999907
Average (Standard dev.)16.667002 (240.935869999999994)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions1515149582
Origin-173.0-169.043.0
Limit1341.01325.0124.0
Spacing1495151582
CellA: 69069.0 Å / B: 69993.0 Å / C: 3788.4001 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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
Number of components: 1

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Component #1: cellular-component, XC (rous sarcoma) cell transiently expressing...

Cellular-componentName: XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2
Recombinant expression: No
SourceSpecies: Rattus (rat)
Source (natural)Organ or tissue: muscle

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Experimental details

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Sample preparation

SpecimenSpecimen state: cell / Method: cryo EM
Sample solutionBuffer solution: DMEM 10% fetal bofine serum / pH: 7.4
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: OTHER

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 50000.0 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: electron tomography / Number of sections: 40
3D reconstructionAlgorithm: BACK PROJECTION / Software: eTomo

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