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

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Basic information

Entry
Database: PDB / ID: 7zg7
TitleStructure of human Apoferritin obtained from ssDNA coated grid
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / apoferritin / ssDNA covered grid / ssDNA
Function / homology
Function and homology information


iron ion sequestering activity / ferritin complex / negative regulation of ferroptosis / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / intracellular sequestering of iron ion / negative regulation of fibroblast proliferation ...iron ion sequestering activity / ferritin complex / negative regulation of ferroptosis / Scavenging by Class A Receptors / Golgi Associated Vesicle Biogenesis / ferroxidase / autolysosome / ferroxidase activity / intracellular sequestering of iron ion / negative regulation of fibroblast proliferation / autophagosome / ferric iron binding / Iron uptake and transport / ferrous iron binding / 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 / membrane / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.77 Å
AuthorsHrebik, D. / Plevka, P.
Funding support Czech Republic, 1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ferritin heavy chain
B: Ferritin heavy chain
C: Ferritin heavy chain
D: Ferritin heavy chain
E: Ferritin heavy chain
F: Ferritin heavy chain
G: Ferritin heavy chain
H: Ferritin heavy chain
I: Ferritin heavy chain
J: Ferritin heavy chain
K: Ferritin heavy chain
L: Ferritin heavy chain
M: Ferritin heavy chain
N: Ferritin heavy chain
O: Ferritin heavy chain
P: Ferritin heavy chain
Q: Ferritin heavy chain
R: Ferritin heavy chain
S: Ferritin heavy chain
T: Ferritin heavy chain
U: Ferritin heavy chain
V: Ferritin heavy chain
W: Ferritin heavy chain
X: Ferritin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)503,032406
Polymers482,79724
Non-polymers20,235382
Water48,2802680
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Ferritin heavy chain / Ferritin H subunit / Cell proliferation-inducing gene 15 protein


Mass: 20116.547 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FTH1, FTH, FTHL6, OK/SW-cl.84, PIG15 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P02794, ferroxidase
#2: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 270 / Source method: obtained synthetically / Formula: Zn
#3: Chemical...
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 112 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2680 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: human heavy chain apoferritin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.02 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPES1
2150 mMsodium chlorideNaCl1
31 mMDTTdithiothreitol1
SpecimenConc.: 4 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: 277.15 K
Details: 6 s blot time,30 s waiting time, ssDNA covered grid

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.08 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3282
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF ChimeraX1.3model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3282
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65786 / Algorithm: BACK PROJECTION / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 7 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 7K3W

7k3w


Accession code: 7K3W / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 7.86 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00436744
ELECTRON MICROSCOPYf_angle_d0.73949464
ELECTRON MICROSCOPYf_dihedral_angle_d14.44114376
ELECTRON MICROSCOPYf_chiral_restr0.045304
ELECTRON MICROSCOPYf_plane_restr0.0056408

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