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- PDB-6sht: Molecular structure of mouse apoferritin resolved at 2.7 Angstrom... -

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Basic information

Entry
Database: PDB / ID: 6sht
TitleMolecular structure of mouse apoferritin resolved at 2.7 Angstroms with the Glacios cryo-microscope
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / Apoferritin / iron binding / iron storing / complex
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation / ferric iron binding / ferrous iron binding / iron ion transport / iron ion binding / immune response / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain ...Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
: / Ferritin heavy chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsHamdi, F. / Tueting, C. / Semchonok, D. / Kyrilis, F. / Meister, A. / Skalidis, I. / Schmidt, L. / Parthier, C. / Stubbs, M.T. / Kastritis, P.L.
CitationJournal: PLoS One / Year: 2020
Title: 2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope.
Authors: Farzad Hamdi / Christian Tüting / Dmitry A Semchonok / Koen M Visscher / Fotis L Kyrilis / Annette Meister / Ioannis Skalidis / Lisa Schmidt / Christoph Parthier / Milton T Stubbs / Panagiotis L Kastritis /
Abstract: Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. ...Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution.
History
DepositionAug 8, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Data collection / Category: em_imaging_optics
Revision 1.2May 22, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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 / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ferritin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,1783
Polymers21,0981
Non-polymers802
Water1,29772
1
A: Ferritin heavy chain
hetero molecules
x 24


Theoretical massNumber of molelcules
Total (without water)508,26772
Polymers506,34324
Non-polymers1,92448
Water43224
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation23
Buried area93330 Å2
ΔGint-682 kcal/mol
Surface area153130 Å2
MethodPISA v1.52 [20/10/2014]

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Components

#1: Protein Ferritin heavy chain / Ferritin H subunit


Mass: 21097.631 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Fth1, Fth / Production host: Escherichia coli (E. coli) / References: UniProt: P09528, ferroxidase
#2: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 72 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Mouse apoferritin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.48 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA / Details: microscope model is Thermofisher Glacios 200 kV
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 1600 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: 30 sec. / Electron dose: 28 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 300 / Details: pixel size was 0.96 Angstroms

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Processing

SoftwareName: PHENIX / Version: 1.9_1692 / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.0.5particle selection
2EPU2.1image acquisition
4Gctf1.0.6CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.9model refinement
10RELION3.0.5initial Euler assignment
11RELION3.0.5final Euler assignment
12RELION3.0.5classification
13RELION3.0.53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 211177
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95733 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 3WNW

3wnw


Accession code: 3WNW / Source name: PDB / Type: experimental model
RefinementResolution: 2.73→245.76 Å / SU ML: 0.33 / Phase error: 24.45 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2543 1731 2.56 %
Rwork0.2509 --
obs0.251 67527 99.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0151513
ELECTRON MICROSCOPYf_angle_d1.5242046
ELECTRON MICROSCOPYf_dihedral_angle_d17.284591
ELECTRON MICROSCOPYf_chiral_restr0.064213
ELECTRON MICROSCOPYf_plane_restr0.006270
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7303-2.81070.48241420.50115389ELECTRON MICROSCOPY100
2.8107-2.90140.39951420.40045381ELECTRON MICROSCOPY100
2.9014-3.00510.33191410.32415408ELECTRON MICROSCOPY100
3.0051-3.12540.24341420.24695391ELECTRON MICROSCOPY100
3.1254-3.26770.19311430.19965411ELECTRON MICROSCOPY100
3.2677-3.440.18651430.17185436ELECTRON MICROSCOPY100
3.44-3.65550.15781430.14265440ELECTRON MICROSCOPY100
3.6555-3.93780.15761440.13615449ELECTRON MICROSCOPY100
3.9378-4.33410.1451440.14855492ELECTRON MICROSCOPY100
4.3341-4.96130.15651450.1575520ELECTRON MICROSCOPY100
4.9613-6.25090.19181480.23925600ELECTRON MICROSCOPY100
6.2509-246.76150.37711540.35055879ELECTRON MICROSCOPY100

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