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

Sci Rep. 2016 Aug 3:6:30909. doi: 10.1038/srep30909.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Glutamate-Ammonia Ligase / chemistry*
  • Maltose-Binding Proteins / chemistry*
  • Protein Multimerization
  • Recombinant Fusion Proteins / chemistry*


  • Maltose-Binding Proteins
  • Recombinant Fusion Proteins
  • Glutamate-Ammonia Ligase