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- EMDB-10205: Molecular structure of mouse apoferritin resolved at 2.7 Angstrom... -

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Basic information

Entry
Database: EMDB / ID: EMD-10205
TitleMolecular structure of mouse apoferritin resolved at 2.7 Angstroms with the Glacios cryo-microscope
Map dataPost-processed map of apoferritin at 200 kV resolved with the FEI Glacios microscope at 2.7 A
Sample
  • Complex: Mouse apoferritinFerritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: MAGNESIUM ION
  • Ligand: water
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 / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / 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 speciesMus musculus (house mouse)
Methodsingle 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 MT / Kastritis PL
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, 2019-
Header (metadata) releaseMay 13, 2020-
Map releaseMay 13, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sht
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6sht
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10205.png

Downloads & links

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Map

FileDownload / File: emd_10205.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map of apoferritin at 200 kV resolved with the FEI Glacios microscope at 2.7 A
Voxel sizeX=Y=Z: 0.96 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.17280681 - 0.30700344
Average (Standard dev.)0.0005604103 (±0.015202475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 245.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.960.960.96
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z245.760245.760245.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1730.3070.001

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Supplemental data

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Mask #1

Fileemd_10205_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post-processed (unmasked) map of apoferritin at 200 kV...

Fileemd_10205_additional.map
AnnotationPost-processed (unmasked) map of apoferritin at 200 kV resolved with the FEI Glacios microscope at 2.7 A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refined half-map of apoferritin at 200 kV resolved...

Fileemd_10205_half_map_1.map
AnnotationRefined half-map of apoferritin at 200 kV resolved with the FEI Glacios microscope
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refined half-map of apoferritin at 200 kV resolved...

Fileemd_10205_half_map_2.map
AnnotationRefined half-map of apoferritin at 200 kV resolved with the FEI Glacios microscope
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse apoferritin

EntireName: Mouse apoferritinFerritin
Components
  • Complex: Mouse apoferritinFerritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Mouse apoferritin

SupramoleculeName: Mouse apoferritin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: Ferritin heavy chain

MacromoleculeName: Ferritin heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 21.097631 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTTASPSQVR QNYHQDAEAA INRQINLELY ASYVYLSMSC YFDRDDVALK NFAKYFLHQS HEEREHAEKL MKLQNQRGGR IFLQDIKKP DRDDWESGLN AMECALHLEK SVNQSLLELH KLATDKNDPH LCDFIETYYL SEQVKSIKEL GDHVTNLRKM G APEAGMAE YLFDKHTLGH GDES

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Macromolecule #2: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 72 / 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: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 1.6 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Detailsmicroscope model is Thermofisher Glacios 200 kV
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 300 / Average exposure time: 30.0 sec. / Average electron dose: 28.0 e/Å2 / Details: pixel size was 0.96 Angstroms
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 211177
CTF correctionSoftware - Name: Gctf (ver. 1.0.6)
Startup modelType of model: EMDB MAP
EMDB ID:

0144


Details: low-pass filtered to 60 Angs
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final 3D classificationNumber classes: 5 / Avg.num./class: 40000 / Software - Name: RELION (ver. 3.0.5)
Details: all classes after 3D reaches resolutions of 4.5 Angs to 6.1 Angs
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.5)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.5) / Number images used: 95733
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3wnw

RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

PDB-6sht:
Molecular structure of mouse apoferritin resolved at 2.7 Angstroms with the Glacios cryo-microscope

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