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- EMDB-3849: Single particle cryo-STEM of ferritin with Fe at low stoichiometry -

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Basic information

Entry
Database: EMDB / ID: EMD-3849
TitleSingle particle cryo-STEM of ferritin with Fe at low stoichiometry
Map dataFerritin with 100 Fe atoms from single particle cryo-STEM data
Sample
  • Complex: Human heavy chain ferritin with Fe at low stoichiometry
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 21.0 Å
AuthorsElad N / Bellapadrona G / Houben L / Sagi I / Elbaum M
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Detection of isolated protein-bound metal ions by single-particle cryo-STEM.
Authors: Nadav Elad / Giuliano Bellapadrona / Lothar Houben / Irit Sagi / Michael Elbaum /
Abstract: Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional ...Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
History
DepositionAug 13, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseOct 18, 2017-
UpdateNov 8, 2017-
Current statusNov 8, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3849.png

Downloads & links

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Map

FileDownload / File: emd_3849.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFerritin with 100 Fe atoms from single particle cryo-STEM data
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.3 Å/pix.
x 80 pix.
= 264. Å		3.3 Å/pix.
x 80 pix.
= 264. Å		3.3 Å/pix.
x 80 pix.
= 264. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.026305879 - 0.04511763
Average (Standard dev.)0.0006757258 (±0.0042300415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 264.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z264.000264.000264.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0260.0450.001

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Supplemental data

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Sample components

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Entire : Human heavy chain ferritin with Fe at low stoichiometry

EntireName: Human heavy chain ferritin with Fe at low stoichiometry
Components
  • Complex: Human heavy chain ferritin with Fe at low stoichiometry

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Supramolecule #1: Human heavy chain ferritin with Fe at low stoichiometry

SupramoleculeName: Human heavy chain ferritin with Fe at low stoichiometry
type: complex / ID: 1 / Parent: 0
Details: Bacterially expressed and purified human heavy chain ferritin mixed with a 100 fold molar excess of Fe(II)SO4
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 504 KDa

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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

Concentration0.13 mg/mL
BufferpH: 7.2 / Component:
ConcentrationFormula
50.0 mMHEPES-NaOH
0.2 MNaCl
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 297 K / Instrument: LEICA EM GP
DetailsFor metal loading, stock protein (2.9 mg/ml) was diluted to 0.25 mg/ml and mixed with a 100 fold molar excess of Fe(II)SO4.

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Electron microscopy

MicroscopeFEI TECNAI F20
DetailsSTEM mode imaging using Fischione model 3000 HAADF detector. Camera length: 520 mm.
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 58 / Average electron dose: 127.0 e/Å2
Details: ADF images collected with a Fischione Model 3000 high-angle annular DF detector
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: OTHER / Cs: 2.0 mm / Nominal magnification: 450000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsFischione 3000 HAADF
Particle selectionNumber selected: 1484 / Details: Manually selected
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2cei


Details: The initial model was prepared from the of the human ferritin heavy chain crystal structure from which all heavy atoms were removed. The protein PDB coordinates were converted to structure ...Details: The initial model was prepared from the of the human ferritin heavy chain crystal structure from which all heavy atoms were removed. The protein PDB coordinates were converted to structure factors and low pass filtered to 4 nm.
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 1484
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)

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