Title | Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. |
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Journal, issue, pages | Sci Rep, Vol. 6, Page 30909, Year 2016 |
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Publish date | Aug 3, 2016 |
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Authors | Francesca Coscia / Leandro F Estrozi / Fabienne Hans / Hélène Malet / Marjolaine Noirclerc-Savoye / Guy Schoehn / Carlo Petosa / |
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PubMed 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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External links | Sci Rep / PubMed:27485862 / PubMed Central |
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Methods | EM (single particle) |
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Resolution | 6.2 Å |
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Structure data | EMDB-4039, PDB-5ldf: Maltose binding protein genetically fused to dodecameric glutamine synthetase Method: EM (single particle) / Resolution: 6.2 Å |
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Source | - Escherichia coli (E. coli)
- salmonella typhi (bacteria)
- escherichia coli o157:h7 (bacteria)
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Keywords | LIGASE / Fusion protein / chimera / dodecamer / symmetrized construct |
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