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- EMDB-55793: 1.80 A cryo-EM structure of Mycobacterium tuberculosis BfrB prepa... -

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Basic information

Entry
Database: EMDB / ID: EMD-55793
Title1.80 A cryo-EM structure of Mycobacterium tuberculosis BfrB prepared under isotope-depleted abundance
Map dataBfrB isotope-depleted - Map
Sample
  • Organelle or cellular component: Mycobacterium tuberculosis BfrB C13 and N15 isotope depleted
    • Protein or peptide: Ferritin BfrB
  • Ligand: water
KeywordsBfrB / Fe storage / C13 and N15 Isotope-depleted / METAL BINDING PROTEIN
Function / homology
Function and homology information


Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / ferroxidase activity / ferric iron binding / peptidoglycan-based cell wall / iron ion transport / ferrous iron binding ...Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / ferroxidase activity / ferric iron binding / peptidoglycan-based cell wall / iron ion transport / ferrous iron binding / intracellular iron ion homeostasis / 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: 1.8 Å
AuthorsHakke SS / Noteborn WEM / Knoops K / Heeren RMA
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)15575 Netherlands
CitationJournal: Anal Chem / Year: 2026
Title: Isotope Decluttering Reduces Spectral Complexity while Maintaining Protein Structure.
Authors: Sneha S Hakke / Willem E M Noteborn / Birol Cabukusta / Ye Gao / Kèvin Knoops / Carmen López-Iglesias / David P A Kilgour / David J Clarke / Anjusha Mathew / Ron M A Heeren /
Abstract: Accurate mass determination is one of the fundamental objectives in mass spectrometry (MS) as it enables confident molecular identification and detection of subtle mass differences. Precise mass ...Accurate mass determination is one of the fundamental objectives in mass spectrometry (MS) as it enables confident molecular identification and detection of subtle mass differences. Precise mass determination typically relies on the measurement of the monoisotopic peak. The increasing number of heavier isotopes, as the molecular mass increases, leads to spectral complexity, broadening of the isotopic distribution, and dispersal of signal intensity, ultimately reducing the signal-to-noise ratio (SNR). These effects are enhanced while analyzing larger proteins and protein complexes. In this study, we expressed and purified two protein complexes, EsxAB and bacterioferritin B (BfrB), under isotope-depleted conditions to reduce the abundances of the heavier isotopes of carbon and nitrogen. We applied isotope depletion to BfrB, which represents the largest mass of the isotope-depleted protein complex studied under native conditions to date, and, for the first time, investigated its structural consequences. Isotope-depleted proteins as well as protein complexes showed simplified mass spectra by reducing the isotopic distribution, with a significant increase in the SNR of the monoisotopic peak followed by improved protein sequence coverage by native top-down MS. Furthermore, our investigation of protein structure, by single-particle analysis using cryo-electron microscopy (cryo-EM), demonstrated that isotope depletion preserves the structural integrity of proteins, even at atomic resolution. Collectively, our findings show that isotope depletion is a suitable method for high-accuracy mass measurement and identification by MS while maintaining the structural integrity of the proteins.
History
DepositionNov 21, 2025-
Header (metadata) releaseMay 20, 2026-
Map releaseMay 20, 2026-
UpdateJun 17, 2026-
Current statusJun 17, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_55793.png

Downloads & links

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Map

FileDownload / File: emd_55793.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBfrB isotope-depleted - Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.51 Å/pix.
x 400 pix.
= 205.2 Å		0.51 Å/pix.
x 400 pix.
= 205.2 Å		0.51 Å/pix.
x 400 pix.
= 205.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.513 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.26170433 - 0.4938398
Average (Standard dev.)0.0002471988 (±0.019691579)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 205.20001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_55793_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BfrB isotope-depleted - Half map B

Fileemd_55793_half_map_1.map
AnnotationBfrB isotope-depleted - Half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BfrB isotope-depleted - Half map A

Fileemd_55793_half_map_2.map
AnnotationBfrB isotope-depleted - Half map A
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 BfrB C13 and N15 isotope depleted

EntireName: Mycobacterium tuberculosis BfrB C13 and N15 isotope depleted
Components
  • Organelle or cellular component: Mycobacterium tuberculosis BfrB C13 and N15 isotope depleted
    • Protein or peptide: Ferritin BfrB
  • Ligand: water

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Supramolecule #1: Mycobacterium tuberculosis BfrB C13 and N15 isotope depleted

SupramoleculeName: Mycobacterium tuberculosis BfrB C13 and N15 isotope depleted
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)

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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: 1833 / 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

Concentration50 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 165000
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

CTF correctionSoftware - Name: cryoSPARC (ver. 4.7.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.7.1) / Number images used: 500000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.7.1)
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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