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Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein

Abstract : Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.
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Contributeur : Frank Thomas <>
Soumis le : lundi 14 novembre 2016 - 10:57:05
Dernière modification le : vendredi 11 décembre 2020 - 14:58:02

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Francesca Coscia, Leandro F Estrozi, Fabienne Hans, Hélène Malet, Marjolaine Noirclerc-Savoye, et al.. Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Scientific Reports, Nature Publishing Group, 2016, 6, pp.30909. ⟨10.1038/srep30909⟩. ⟨hal-01396217⟩



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