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- EMDB-14679: Tribolium castaneum hexamerin 2 -

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Basic information

Entry
Database: EMDB / ID: EMD-14679
TitleTribolium castaneum hexamerin 2
Map datahexamerin main map
Sample
  • Complex: Hexamerin 2
    • Protein or peptide: Larval serum protein 1 gamma chain-like Protein
Function / homology
Function and homology information


Arthropod hemocyanins / insect LSPs signature 2. / Hemocyanin, N-terminal / Hemocyanin, N-terminal domain superfamily / Hemocyanin, all-alpha domain / Hemocyanin, C-terminal domain superfamily / Hemocyanin/hexamerin middle domain / Hemocyanin, C-terminal / Hemocyanin, copper containing domain / Hemocyanin, ig-like domain / Hemocyanin/hexamerin ...Arthropod hemocyanins / insect LSPs signature 2. / Hemocyanin, N-terminal / Hemocyanin, N-terminal domain superfamily / Hemocyanin, all-alpha domain / Hemocyanin, C-terminal domain superfamily / Hemocyanin/hexamerin middle domain / Hemocyanin, C-terminal / Hemocyanin, copper containing domain / Hemocyanin, ig-like domain / Hemocyanin/hexamerin / Di-copper centre-containing domain superfamily / Immunoglobulin E-set
Similarity search - Domain/homology
Larval serum protein 1 gamma chain-like Protein
Similarity search - Component
Biological speciesTribolium castaneum (red flour beetle) / red flour beetle (red flour beetle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsValentova L / Fuzik T / Plevka P
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-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14679.png

Downloads & links

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Map

FileDownload / File: emd_14679.map.gz / Format: CCP4 / Size: 23 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhexamerin main map
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 2.4
Minimum - Maximum-7.518139 - 11.22593
Average (Standard dev.)-2.9057543e-09 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-91-91-91
Dimensions182182182
Spacing182182182
CellA=B=C: 149.604 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: mask of the half maps

Fileemd_14679_additional_1.map
Annotationmask of the half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14679_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14679_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
    • Protein or peptide: Larval serum protein 1 gamma chain-like Protein

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Supramolecule #1: Hexamerin 2

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

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Macromolecule #1: Larval serum protein 1 gamma chain-like Protein

MacromoleculeName: Larval serum protein 1 gamma chain-like Protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: red flour beetle (red flour beetle)
Molecular weightTheoretical: 84.656922 KDa
SequenceString: MRFILVAVLA GLCAVSAIST SESSNYSQQQ RDVWRLFKYI NQPSYYKDHV EIAHSYYFYD HASNYAKHEV VEEFYRYFKY DTFLQRGEI FSVFHGEHLK QAIALFKLFY YANDFDTFYK TAVWARQHVN EGMFLYAFSV ALIHRPDTYY FSLPPIYEIY P HYFYNYEV ...String:
MRFILVAVLA GLCAVSAIST SESSNYSQQQ RDVWRLFKYI NQPSYYKDHV EIAHSYYFYD HASNYAKHEV VEEFYRYFKY DTFLQRGEI FSVFHGEHLK QAIALFKLFY YANDFDTFYK TAVWARQHVN EGMFLYAFSV ALIHRPDTYY FSLPPIYEIY P HYFYNYEV IQKAQHYKQM YYGQDGAHYN DRTIYANYSG YYVNVYPEQA LAYFTEDVGV NSFYYYYNLY YPYWMSGEEF NL KYDNRGE IFYYMYQQIL ARYYLERLSH GFGEIDHFDW EVPFESGYYP SMCYPNGLYF PTRHAYAHLY EYFYNYGQHY GFN KYAHSY THISDYERRI HDVIDSGYVH THSGQKVDLF SHEGLDILGN LIEGNPESPY YHYYGAYQVF ARHLLGYSHQ PLTF HKLHP SALEHFETSM RDPAFYQLYK KLLGFFFRYK SQHYHYYDEH DLAYHGVHVK HVEVDPLVTY FDYFYADLSN AVYVT PEEF VHDSFKVHVA QERLNHKPFT YKIYIDSDKD TEAVVKVFLG PKYDEYGRYI NLTENWMNFV QFDHFVYKLK SGENVI SRN SHEIYNYIHD RTSYYELYQK AFGVYNKDQH FQFHDNQFFF GFPQRYMLPR GSPEGMTYQF YVFVTKYHPY KAHASVP MV GSGMHYVDAY PMGYPFDRPV YYEELFYALP NSYFQDVRIY YQGHNYEHHA NHLIEM

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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

Concentration5 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 / Pretreatment - Type: GLOW DISCHARGE
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: 165000
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: 1711 / Average exposure time: 5.0 sec. / Average electron dose: 57.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 16616
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Software - details: ctf estimation
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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 3106
FSC plot (resolution estimation)

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Atomic model buiding 1

Detailsreal space refine
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 45.57 / Target criteria: corelation coefficient
Output model

PDB-7ze1:
Tribolium castaneum hexamerin 2

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