title: 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.
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Francesca
authors.initials: F
authors.lastname: Coscia
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Leandro F
authors.initials: LF
authors.lastname: Estrozi
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Hans
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Malet
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Noirclerc-Savoye
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Guy
authors.initials: G
authors.lastname: Schoehn
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, 38044 Grenoble, France.
authors.forename: Carlo
authors.initials: C
authors.lastname: Petosa
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry*; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D005974:Glutamate-Ammonia Ligase