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

Coscia, Francesca
Estrozi, Leandro F
Hans, Fabienne
Malet, Hélène
Noirclerc-Savoye, Marjolaine
Schoehn, Guy
Petosa, Carlo

Open link

Publication date

August 2016

DOI

10.1038/srep30909

Publisher

Springer Science and Business Media LLC

Abstract

International audienceRecent 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 E...