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- EMDB-11638: Cryo-EM structure of mouse heavy-chain apoferritin at 1.22 A -

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Basic information

Entry
Database: EMDB / ID: EMD-11638
TitleCryo-EM structure of mouse heavy-chain apoferritin at 1.22 A
Map data
Sample
  • Complex: Mouse heavy-chain apoferritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: ZINC ION
  • Ligand: water
KeywordsIron storage / metal binding protein
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation / ferric iron binding / autophagosome / ferrous iron binding / iron ion transport / intracellular iron ion homeostasis / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / membrane / cytosol
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: 1.22 Å
AuthorsNakane T / Kotecha A
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1012 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1013 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z) United Kingdom
CitationJournal: Nature / Year: 2020
Title: Single-particle cryo-EM at atomic resolution.
Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / ...Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / Tomasz Uchański / Lingbo Yu / Dimple Karia / Evgeniya V Pechnikova / Erwin de Jong / Jeroen Keizer / Maarten Bischoff / Jamie McCormack / Peter Tiemeijer / Steven W Hardwick / Dimitri Y Chirgadze / Garib Murshudov / A Radu Aricescu / Sjors H W Scheres /
Abstract: The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical ...The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA receptor homopentamer. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
History
DepositionAug 20, 2020-
Header (metadata) releaseOct 28, 2020-
Map releaseOct 28, 2020-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing 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-7a4m
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7a4m
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11638.png

Downloads & links

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Map

FileDownload / File: emd_11638.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.53 Å/pix.
x 256 pix.
= 136.499 Å		0.53 Å/pix.
x 256 pix.
= 136.499 Å		0.53 Å/pix.
x 256 pix.
= 136.499 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.116 / Movie #1: 0.05
Minimum - Maximum-0.15899505 - 0.52106285
Average (Standard dev.)-0.0007289657 (±0.02642681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 136.4992 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.533199218750.533199218750.53319921875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z136.499136.499136.499
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1590.521-0.001

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Supplemental data

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Mask #1

Fileemd_11638_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11638_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11638_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse heavy-chain apoferritin

EntireName: Mouse heavy-chain apoferritin
Components
  • Complex: Mouse heavy-chain apoferritin
    • Protein or peptide: Ferritin heavy chain
  • Ligand: FE (III) ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: Mouse heavy-chain apoferritin

SupramoleculeName: Mouse heavy-chain apoferritin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 500 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: 20.079594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
PSQVRQNYHQ DAEAAINRQI NLELYASYVY LSMSCYFDRD DVALKNFAKY FLHQSHEERE HAEKLMKLQN QRGGRIFLQD IKKPDRDDW ESGLNAMECA LHLEKSVNQS LLELHKLATD KNDPHLCDFI ETYYLSEQVK SIKELGDHVT NLRKMGAPEA G MAEYLFDK HTLG

UniProtKB: Ferritin heavy chain

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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: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 110 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

BufferpH: 7.5 / Details: 20mM HEPES pH 7.5 150mM NaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 270000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

9865

Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 363126
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-7a4m:
Cryo-EM structure of mouse heavy-chain apoferritin at 1.22 A

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