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- PDB-9tcm: 1.79 A cryo-EM structure of Mycobacterium tuberculosis BfrB prepa... -

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Basic information

Entry
Database: PDB / ID: 9tcm
Title1.79 A cryo-EM structure of Mycobacterium tuberculosis BfrB prepared under natural isotope abundance
ComponentsFerritin BfrB
KeywordsMETAL BINDING PROTEIN / BfrB / Fe storage / C13 and N15 Isotope-depleted
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.79 Å
AuthorsHakke, S.S. / Noteborn, W.E.M. / Knoops, K. / Heeren, R.M.A.
Funding support Netherlands, 1items
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, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 20, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ferritin BfrB
B: Ferritin BfrB
C: Ferritin BfrB
D: Ferritin BfrB
E: Ferritin BfrB
F: Ferritin BfrB
G: Ferritin BfrB
H: Ferritin BfrB
I: Ferritin BfrB
J: Ferritin BfrB
K: Ferritin BfrB
L: Ferritin BfrB
M: Ferritin BfrB
N: Ferritin BfrB
O: Ferritin BfrB
P: Ferritin BfrB
Q: Ferritin BfrB
R: Ferritin BfrB
S: Ferritin BfrB
T: Ferritin BfrB
V: Ferritin BfrB
W: Ferritin BfrB
X: Ferritin BfrB
Y: Ferritin BfrB


Theoretical massNumber of molelcules
Total (without water)491,13424
Polymers491,13424
Non-polymers00
Water33,8861881
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Ferritin BfrB / Non-heme ferritin Ftn / Nox19


Mass: 20463.936 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: bfrB / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WNE5, ferroxidase
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1881 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mycobacterium tuberculosis BfrB normal isotope distribution
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 50 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 285 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.7.1particle selection
2EPUimage acquisition
4cryoSPARC4.7.1CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARC4.7.1initial Euler assignment
10cryoSPARC4.7.1final Euler assignment
11cryoSPARC4.7.1classification
12cryoSPARC4.7.13D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500000 / Symmetry type: POINT
RefinementHighest resolution: 1.79 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00233000
ELECTRON MICROSCOPYf_angle_d0.41944640
ELECTRON MICROSCOPYf_dihedral_angle_d3.3414392
ELECTRON MICROSCOPYf_chiral_restr0.0344896
ELECTRON MICROSCOPYf_plane_restr0.0036024

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