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- EMDB-10466: Cryo-ET on GroEL -

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Basic information

Entry
Database: EMDB / ID: EMD-10466
TitleCryo-ET on GroEL
Map dataReconstructed tomography data.The GroEL specimen preparation method is Preassis, and a Quantifoil continuous carbon film grid (R2/2) was used. In the hole of this tomogram, the carbon film was broken.
Sample
  • Complex: GroEL
Biological speciesEscherichia coli K-12 (bacteria)
Methodelectron tomography / cryo EM
AuthorsZou XD / Zhao JJ / Xu HY / Carroni M
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council2012.0112 Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nat Commun / Year: 2021
Title: A simple pressure-assisted method for MicroED specimen preparation.
Authors: Jingjing Zhao / Hongyi Xu / Hugo Lebrette / Marta Carroni / Helena Taberman / Martin Högbom / Xiaodong Zou /
Abstract: Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a ...Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.
History
DepositionNov 9, 2019-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing 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_10466.png

Downloads & links

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Map

FileDownload / File: emd_10466.map.gz / Format: CCP4 / Size: 297.4 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationReconstructed tomography data.The GroEL specimen preparation method is Preassis, and a Quantifoil continuous carbon film grid (R2/2) was used. In the hole of this tomogram, the carbon film was broken.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.8 Å/pix.
x 175 pix.
= 2415. Å		13.8 Å/pix.
x 928 pix.
= 12806.4 Å		13.8 Å/pix.
x 960 pix.
= 13248. 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: 13.8 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-2171.0 - 2096.0
Average (Standard dev.)105.888145 (±116.60059)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-161788
Dimensions928960175
Spacing960928175
CellA: 13248.0 Å / B: 12806.4 Å / C: 2415.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z13.813.813.8
M x/y/z960928175
origin x/y/z0.0000.0000.000
length x/y/z13248.00012806.4002415.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS17-1688
NC/NR/NS960928175
D min/max/mean-2171.0002096.000105.888

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Supplemental data

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Additional map: Reconstructed tomography data.The GroEL specimen preparation method is...

Fileemd_10466_additional_1.map
AnnotationReconstructed tomography data.The GroEL specimen preparation method is Preassis, and a Quantifoil continuous carbon film grid (R2/2) was used. In the hole of this tomogram, the carbon film was intact.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw tomography data. The GroEL specimen preparation method...

Fileemd_10466_additional_2.map
AnnotationRaw tomography data. The GroEL specimen preparation method is Preassis, and a Quantifoil continuous carbon film grid (R2/2) was used. In the hole of this tomogram, the carbon film was intact.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw tomography data. The GroEL specimen preparation method...

Fileemd_10466_additional_3.map
AnnotationRaw tomography data. The GroEL specimen preparation method is Preassis, and a Quantifoil continuous carbon film grid (R2/2) was used. In the hole of this tomogram, the carbon film was broken.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GroEL

EntireName: GroEL
Components
  • Complex: GroEL

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Supramolecule #1: GroEL

SupramoleculeName: GroEL / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightExperimental: 0.58 kDa/nm

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.70 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.2 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Details: A new freezing method was used, called Preassis.
DetailsThe GroEL grid for cryoET data collection was prepared by a new specimen preparation method, called Preassis. A Quantifoil continuous carbon grid was used.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 50

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