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- EMDB-14705: Structure of human Apoferritin obtained from ssDNA coated grid -

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Basic information

Entry
Database: EMDB / ID: EMD-14705
TitleStructure of human Apoferritin obtained from ssDNA coated grid
Map dataFinal Map
Sample
  • Complex: human heavy chain apoferritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: ZINC ION
  • Ligand: SODIUM IONSodium
  • Ligand: water
Function / homology
Function and homology information


iron ion sequestering activity / intracellular ferritin complex / autolysosome / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding ...iron ion sequestering activity / intracellular ferritin complex / autolysosome / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / ferric iron binding / ferrous iron binding / Iron uptake and transport / tertiary granule lumen / iron ion transport / intracellular iron ion homeostasis / ficolin-1-rich granule lumen / immune response / iron ion binding / negative regulation of cell population proliferation / Neutrophil degranulation / extracellular exosome / extracellular region / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.77 Å
AuthorsHrebik D / Plevka P
Funding support Czech Republic, 1 items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic 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
DepositionApr 2, 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_14705.png

Downloads & links

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Map

FileDownload / File: emd_14705.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal Map
Voxel sizeX=Y=Z: 0.4525 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.054408703 - 0.09981596
Average (Standard dev.)0.00037432782 (±0.009038803)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 126.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 1

Fileemd_14705_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_14705_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human heavy chain apoferritin

EntireName: human heavy chain apoferritin
Components
  • Complex: human heavy chain apoferritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: ZINC ION
  • Ligand: SODIUM IONSodium
  • Ligand: water

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Supramolecule #1: human heavy chain apoferritin

SupramoleculeName: human heavy chain apoferritin / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 20 KDa

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Macromolecule #1: Ferritin heavy chain

MacromoleculeName: Ferritin heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.116547 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
TSQVRQNYHQ DSEAAINRQI NLELYASYVY LSMSYYFDRD DVALKNFAKY FLHQSHEERE HAEKLMKLQN QRGGRIFLQD IKKPDCDDW ESGLNAMECA LHLEKNVNQS LLELHKLATD KNDPHLCDFI ETHYLNEQVK AIKELGDHVT NLRKMGAPES G LAEYLFDK HTLG

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 270 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 112
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 2680 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration4.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
30.0 mMHEPES
150.0 mMsodium chlorideNaClSodium chloride
1.0 mMDTTdithiothreitol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: NITROGEN
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 6 s blot time,30 s waiting time, ssDNA covered grid.

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Electron microscopy

MicroscopeFEI TITAN 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: 1.7 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 3282 / Average exposure time: 4.08 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3282
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

22658

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 1.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 65786
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7k3w

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 7 / Target criteria: correlation coefficient
Output model

PDB-7zg7:
Structure of human Apoferritin obtained from ssDNA coated grid

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