Fusion to a symmetric scaffold allows cryo‐ EM analysis of a small monomeric protein

Abstract

Single particle cryo‐electron microscopy (cryoEM) is a powerful method for determining the structures of large macromolecules and their complexes. Recent technical advances in direct electron detectors, phase plates and computational procedures for image processing have revolutionized the field, allowing 3D reconstructions to be obtained at near‐atomic resolution (<4 Å). However, most monomeric proteins of biomedical interest remain too small (<100 kDa) for cryo‐EM analysis. In this work we propose a new method to overcome this size limitation by fusing a monomeric protein (target) to a homo‐oligomeric scaffold protein, whose large size and symmetry facilitates cryoEM analysis. As proof‐of‐principle, we fused maltose‐binding protein (MBP), a 40 kDa monomer, to glutamine synthetase (GS), a dodecamer formed by two hexameric rings. We designed an initial MBP‐GS construct with a linker sufficiently long to allow the fused moieties to fold properly without mutual steric hindrance. The junction was optimized by progressively deleting linker and/or flanking residues to reduce the spatial separation between the two fusion partners, thereby restricting the relative mobility of the target subunits (Figure A). We bacterially expressed 18 MBP‐GS constructs and used biophysical analysis (native poly‐acrylamide gel electrophoresis, size exclusion chromatography, dynamic light scattering and differential scanning fluorimetry) to screen for particle compactness and homogeneity. Negative stain EM analysis confirmed the trend observed by the biophysical assays, and both approaches identified the same construct as the most compact and conformationally homogeneous. Cryo‐EM analysis of the most promising construct led to a reconstruction with an overall resolution of 4.2 Å (FSC=0.143 criterion) and a local resolution that varied from ~4 Å within the GS subunit to between 6 and 10 Å within the MBP subunit. Fitting the GS crystal structure into this map revealed an excellent fit, allowing all secondary structure elements and certain large side chains to be visualized. A good fit was also observed for MBP: the N‐ and C‐terminal lobes enclosing the active site were clearly defined, several helical elements were well resolved, and the bound maltose ligand was partly visible (Figure B). These findings illustrate the feasibility of using homo‐oligomeric scaffolds to enable cryo‐EM analysis of monomeric proteins, raising the prospect of applying this strategy to more challenging structures resistant to crystallographic and NMR analysis.