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- PDB-8cpt: Human apoferritin after 488 nm laser exposure -

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Basic information

Entry
Database: PDB / ID: 8cpt
TitleHuman apoferritin after 488 nm laser exposure
ComponentsFerritin heavy chain, N-terminally processed
KeywordsMETAL BINDING PROTEIN / Iron binding protein
Function / homology
Function and homology information


iron ion sequestering activity / : / 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 / : / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.79 Å
AuthorsLast, M.G.F. / Noteborn, W.E.M. / Sharp, T.H.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)VI.Vidi.193.014 Netherlands
CitationJournal: J Struct Biol / Year: 2023
Title: Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM.
Authors: Mart G F Last / Willem E M Noteborn / Lenard M Voortman / Thomas H Sharp /
Abstract: Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A ...Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A concern in this combined method (called SR-cryoCLEM), however, is whether and how fluorescence imaging prior to cryoEM acquisition is detrimental to sample integrity. In this report, we investigated the effect of high-dose laser light (405, 488, and 561 nm) irradiation on apoferritin samples prepared for cryoEM with excitation wavelengths commonly used in fluorescence microscopy, and compared these samples to controls that were kept in the dark. We found that laser illumination, of equal duration and intensity as used in cryo-single molecule localization microscopy (cryoSMLM) and in the presence of high concentrations of fluorescent protein, did not affect the achievable resolution in cryoEM, with final reconstructions reaching resolutions of ∼ 1.8 Å regardless of the laser illumination. The finding that super-resolution fluorescence imaging of cryosamples prior to cryoEM data acquisition does not limit the achievable resolution suggests that super-resolution cryo-fluorescence microscopy and in situ structural biology using cryoEM are entirely compatible.
History
DepositionMar 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_3d_fitting_list
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2Nov 22, 2023Group: Database references / Category: citation / citation_author / Item: _citation.journal_volume / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: Ferritin heavy chain, N-terminally processed
2: Ferritin heavy chain, N-terminally processed
4: Ferritin heavy chain, N-terminally processed
6: Ferritin heavy chain, N-terminally processed
A: Ferritin heavy chain, N-terminally processed
B: Ferritin heavy chain, N-terminally processed
E: Ferritin heavy chain, N-terminally processed
F: Ferritin heavy chain, N-terminally processed
G: Ferritin heavy chain, N-terminally processed
H: Ferritin heavy chain, N-terminally processed
I: Ferritin heavy chain, N-terminally processed
K: Ferritin heavy chain, N-terminally processed
M: Ferritin heavy chain, N-terminally processed
O: Ferritin heavy chain, N-terminally processed
P: Ferritin heavy chain, N-terminally processed
Q: Ferritin heavy chain, N-terminally processed
S: Ferritin heavy chain, N-terminally processed
U: Ferritin heavy chain, N-terminally processed
W: Ferritin heavy chain, N-terminally processed
X: Ferritin heavy chain, N-terminally processed
Y: Ferritin heavy chain, N-terminally processed
a: Ferritin heavy chain, N-terminally processed
e: Ferritin heavy chain, N-terminally processed
r: Ferritin heavy chain, N-terminally processed
hetero molecules


Theoretical massNumber of molelcules
Total (without water)486,13862
Polymers485,24624
Non-polymers89238
Water34,3371906
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area96420 Å2
ΔGint-753 kcal/mol
Surface area141270 Å2

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Components

#1: Protein ...
Ferritin heavy chain, N-terminally processed


Mass: 20218.570 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 (E. coli) / References: UniProt: P02794
#2: Chemical...
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Na
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1906 / 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: ApoferritinFerritin / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.484 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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: 130000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.4/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Windows / Type: package
EM software
IDNameVersionCategory
1RELION4particle selection
2EPU2.10.0image acquisition
4RELION4CTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 1.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9700 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6Z6U

6z6u


Accession code: 6Z6U / Source name: PDB / Type: experimental model

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