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

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

doi:10.1038/srep30909

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.

Highlights

The core protein in which green fluorescent protein (GFP, 27 kDa) was inserted within a loop on the HBV capsid surface
By fusing monomeric maltose-binding protein (MBP) to dodecameric glutamine synthetase (GS) and progressively deleting residues in the junction, we generated a series of MBP-GS constructs that assembled as chimeric particles sufficiently large for cryo-EM analysis
Biophysical assays combined with negative-stain EM identified the two most promising MBP-GS constructs (Δ2 and Δ5)

Summary

Introduction

The core protein in which green fluorescent protein (GFP, 27 kDa) was inserted within a loop on the HBV capsid surface. We show that by fusing a monomeric protein to a homo-oligomeric scaffold and screening different-sized junction regions, a suitably rigid chimeric particle can be generated whose large size and high symmetry facilitates cryo-EM analysis, resulting in a 3D reconstruction of the target structure at sub-nanometre resolution. We anticipate that this strategy will prove a useful addition to the toolkit of methods available for investigating monomeric protein structures resistant to NMR and crystallographic analysis

Methods

Results

Conclusion