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- EMDB-12738: 2.12 A cryo-EM structure of Mycobacterium tuberculosis Ferritin -

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Basic information

Entry
Database: EMDB / ID: EMD-12738
Title2.12 A cryo-EM structure of Mycobacterium tuberculosis Ferritin
Map dataSharpened map with Locspiral.
Sample
  • Organelle or cellular component: Mycobacterium tuberculosis ferritin
    • Protein or peptide: Ferritin BfrB
  • Ligand: water
KeywordsIron storage / Ferroxidase / Bacterial Ferritin / Octahedral symmetry. / METAL TRANSPORT
Function / homology
Function and homology information


Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / peptidoglycan-based cell wall / ferric iron binding / ferrous iron binding ...Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / peptidoglycan-based cell wall / ferric iron binding / ferrous iron binding / iron ion transport / response to hypoxia / extracellular region / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Ferritin, prokaryotic-type / 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
Bacterioferritin BfrB
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsGijsbers A / Zhang Y
Funding supportEuropean Union, Netherlands, 2 items
OrganizationGrant numberCountry
European Union (EU)766970European Union
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2021
Title: Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development.
Authors: Abril Gijsbers / Yue Zhang / Ye Gao / Peter J Peters / Raimond B G Ravelli /
Abstract: The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol ...The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Å resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164-181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.
History
DepositionApr 10, 2021-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
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: 7
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7o6e
  • Surface level: 7
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7o6e
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12738.png

Downloads & links

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Map

FileDownload / File: emd_12738.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map with Locspiral.
Voxel sizeX=Y=Z: 0.6514 Å
Density
Contour LevelBy AUTHOR: 7.0 / Movie #1: 7
Minimum - Maximum0.0 - 25.939672000000002
Average (Standard dev.)0.4104235 (±1.6182348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 208.448 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.65140.65140.6514
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z208.448208.448208.448
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean0.00025.9400.410

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Supplemental data

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Mask #1

Fileemd_12738_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Relion postprocessing sharpened map

Fileemd_12738_additional_1.map
AnnotationRelion postprocessing sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12738_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12738_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mycobacterium tuberculosis ferritin

EntireName: Mycobacterium tuberculosis ferritin
Components
  • Organelle or cellular component: Mycobacterium tuberculosis ferritin
    • Protein or peptide: Ferritin BfrB
  • Ligand: water

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Supramolecule #1: Mycobacterium tuberculosis ferritin

SupramoleculeName: Mycobacterium tuberculosis ferritin / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Mycobacterium tuberculosis ferritin
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: H37Rv
Molecular weightTheoretical: 490 KDa

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Macromolecule #1: Ferritin BfrB

MacromoleculeName: Ferritin BfrB / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 20.463936 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MTEYEGPKTK FHALMQEQIH NEFTAAQQYV AIAVYFDSED LPQLAKHFYS QAVEERNHAM MLVQHLLDRD LRVEIPGVDT VRNQFDRPR EALALALDQE RTVTDQVGRL TAVARDEGDF LGEQFMQWFL QEQIEEVALM ATLVRVADRA GANLFELENF V AREVDVAP AASGAPHAAG GRL

UniProtKB: Bacterioferritin BfrB

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 1392 / 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

Concentration11 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris
150.0 mMNaClSodium Chloride
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 2000 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 93.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsBasic direct alignments were done as well as astigmatism and coma alignment using AutoCTF
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2518 / Average exposure time: 1.3 sec. / Average electron dose: 40.0 e/Å2
Details: Images were acquired in superresolution counting mode at a speed of 64 movies/hour and stored as tiff lzw non-gain normalised
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.2 µm / Calibrated magnification: 76757 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 130000
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

Particle selectionNumber selected: 249563
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3qd8

Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 163568
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 40 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3qd8


Chain - Chain ID: A / Chain - Residue range: 10-181 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 28.1 / Target criteria: Correlation coefficient
Output model

PDB-7o6e:
2.12 A cryo-EM structure of Mycobacterium tuberculosis Ferritin

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