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- PDB-7ze1: Tribolium castaneum hexamerin 2 -

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Basic information

Entry
Database: PDB / ID: 7ze1
TitleTribolium castaneum hexamerin 2
ComponentsLarval serum protein 1 gamma chain-like Protein
KeywordsUNKNOWN FUNCTION / hexamerin / tribolium castaneum / storage 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsValentova, L. / Fuzik, T. / Plevka, P.
Funding support Czech Republic, European Union, 4items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Larval serum protein 1 gamma chain-like Protein


Theoretical massNumber of molelcules
Total (without water)84,6571
Polymers84,6571
Non-polymers00
Water0
1
A: Larval serum protein 1 gamma chain-like Protein
x 6


Theoretical massNumber of molelcules
Total (without water)507,9426
Polymers507,9426
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
MethodUCSF CHIMERA

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Components

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


Mass: 84656.922 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tribolium castaneum (red flour beetle) / References: UniProt: D6WUQ9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Hexamerin 2 / Type: COMPLEX / Details: hexamerin isolated from Tribolium castaneum pupae / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.52 MDa / Experimental value: NO
Source (natural)Organism: Tribolium castaneum (red flour beetle)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
130 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2100 mMpotassium chlorideKCl1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 57 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1711
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4Gctf1.06CTF correctionctf estimation
7Coot0.9.5model fitting
9PHENIX1.19model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 16616
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3106 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 45.57 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: corelation coefficient / Details: real space refine
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035917
ELECTRON MICROSCOPYf_angle_d0.4928045
ELECTRON MICROSCOPYf_dihedral_angle_d4.235777
ELECTRON MICROSCOPYf_chiral_restr0.045743
ELECTRON MICROSCOPYf_plane_restr0.0041039

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