9TCN

1.80 A cryo-EM structure of Mycobacterium tuberculosis BfrB prepared under isotope-depleted abundance

Summary for 9TCN

• Entry DOI: 10.2210/pdb9tcn/pdb
• EMDB information: 55793
• Descriptor: Ferritin BfrB (2 entities in total)
• Functional Keywords: bfrb, fe storage, c13 and n15 isotope-depleted, metal binding protein
• Biological source: Mycobacterium tuberculosis H37Rv
• Total number of polymer chains: 24
• Total formula weight: 491134.46

Authors

Hakke, S.S.,Noteborn, W.E.M.,Knoops, K.,Heeren, R.M.A. (deposition date: 2025-11-21, release date: 2026-05-20, Last modification date: 2026-06-17)

Primary citation

Hakke, S.S.,Noteborn, W.E.M.,Cabukusta, B.,Gao, Y.,Knoops, K.,Lopez-Iglesias, C.,Kilgour, D.P.A.,Clarke, D.J.,Mathew, A.,Heeren, R.M.A.
Isotope Decluttering Reduces Spectral Complexity while Maintaining Protein Structure.
Anal.Chem., 98:16063-16072, 2026

PubMed 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 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.

PubMed: 42212855
DOI: 10.1021/acs.analchem.5c08046

Experimental method

ELECTRON MICROSCOPY (1.8 Å)