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- EMDB-28259: Mouse apoferritin heavy chain with zinc determined using single-p... -

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Basic information

Entry
Database: EMDB / ID: EMD-28259
TitleMouse apoferritin heavy chain with zinc determined using single-particle cryo-EM with Apollo camera.
Map data
Sample
  • Complex: Mouse apoferritin heavy chain
    • Protein or peptide: Ferritin heavy chain, N-terminally processed
  • Ligand: ZINC ION
  • Ligand: FE (III) ION
  • Ligand: water
Keywordsbind with iron / METAL BINDING PROTEIN
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome / 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: 1.66 Å
AuthorsPeng R / Fu X / Mendez JH / Randolph PH / Bammes B / Stagg SM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143805 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139616 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM119032 United States
CitationJournal: J Struct Biol X / Year: 2023
Title: Characterizing the resolution and throughput of the Apollo direct electron detector.
Authors: Ruizhi Peng / Xiaofeng Fu / Joshua H Mendez / Peter S Randolph / Benjamin E Bammes / Scott M Stagg /
Abstract: Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been ...Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.
History
DepositionSep 28, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28259.png

Downloads & links

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Map

FileDownload / File: emd_28259.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.6 Å/pix.
x 480 pix.
= 287.52 Å		0.6 Å/pix.
x 480 pix.
= 287.52 Å		0.6 Å/pix.
x 480 pix.
= 287.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.599 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06
Minimum - Maximum-0.11750294 - 0.25829893
Average (Standard dev.)0.00006163014 (±0.006385368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 287.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28259_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: #2

Fileemd_28259_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_28259_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Mouse apoferritin heavy chain

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

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Supramolecule #1: Mouse apoferritin heavy chain

SupramoleculeName: Mouse apoferritin heavy chain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 506 KDa

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Macromolecule #1: Ferritin heavy chain, N-terminally processed

MacromoleculeName: Ferritin heavy chain, N-terminally processed / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
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: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 24 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 2975 / 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

Concentration4.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClsodium chloride
1.0 mMDTTDL-Dithiothreitol

Details: DTT are added freshly before use.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
SoftwareName: Leginon (ver. 3.3)
Details: leginon are used for automated single particle data collection.
Image recordingFilm or detector model: DIRECT ELECTRON APOLLO (4k x 4k) / Digitization - Dimensions - Width: 8192 pixel / Digitization - Dimensions - Height: 8192 pixel / Number grids imaged: 1 / Number real images: 3804 / Average exposure time: 1.597 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 80.0 µm / Calibrated magnification: 72621 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 75000
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

DetailsDirect Electron Apollo
Particle selectionNumber selected: 183073
Startup modelType of model: INSILICO MODEL / In silico model: ab-initial model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 286970
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 78107 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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