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- EMDB-14680: Tribolium castaneum hexamerin 2 from aggregated sample -

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Basic information

Entry
Database: EMDB / ID: EMD-14680
TitleTribolium castaneum hexamerin 2 from aggregated sample
Map datahexamerin main map
Sample
  • Complex: Hexamerin 2
Keywordshexamerin / tribolium castaneum / storage protein / UNKNOWN FUNCTION
Biological speciesTribolium castaneum (red flour beetle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsValentova L / Plevka P / Fuzik T
Funding support Czech Republic, European Union, 4 items
OrganizationGrant numberCountry
Czech Science FoundationGX19-25982X Czech Republic
European Regional Development FundCZ.1.05/1.1.00/02.0070European Union
Ministry of Education (MoE, Czech Republic)LM2011033 Czech Republic
Ministry of Education (MoE, Czech Republic)LM2015043 Czech Republic
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules.
Authors: Dominik Hrebík / Mária Gondová / Lucie Valentová / Tibor Füzik / Antonín Přidal / Jiří Nováček / Pavel Plevka /
Abstract: Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, ...Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air-water interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.
History
DepositionMar 30, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14680.png

Downloads & links

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Map

FileDownload / File: emd_14680.map.gz / Format: CCP4 / Size: 23 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhexamerin main map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 182 pix.
= 189.28 Å		1.04 Å/pix.
x 182 pix.
= 189.28 Å		1.04 Å/pix.
x 182 pix.
= 189.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.4
Minimum - Maximum-10.837232999999999 - 17.353280000000002
Average (Standard dev.)0.000000000703549 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-91-91-91
Dimensions182182182
Spacing182182182
CellA=B=C: 189.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: mask for half maps

Fileemd_14680_additional_1.map
Annotationmask for half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14680_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14680_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexamerin 2

EntireName: Hexamerin 2
Components
  • Complex: Hexamerin 2

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Supramolecule #1: Hexamerin 2

SupramoleculeName: Hexamerin 2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: hexamerin isolated from Tribolium castaneum pupae
Source (natural)Organism: Tribolium castaneum (red flour beetle)
Molecular weightTheoretical: 520 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
100.0 mMKClpotassium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 1514 / Average exposure time: 5.0 sec. / Average electron dose: 63.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 51895
Startup modelType of model: OTHER / Details: stochastic gradient descent
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 10362
FSC plot (resolution estimation)

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