5LDF

Maltose binding protein genetically fused to dodecameric glutamine synthetase

Summary for 5LDF

• Entry DOI: 10.2210/pdb5ldf/pdb
• EMDB information: 4039
• Related PRD ID: PRD_900001
• Descriptor: Glutamine synthetase, Maltose-binding periplasmic protein, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose (3 entities in total)
• Functional Keywords: fusion protein, chimera, dodecamer, symmetrized construct, ligase
• Biological source: Salmonella typhi; More
• Total number of polymer chains: 24
• Total formula weight: 1112180.58

Authors

Coscia, F.,Petosa, C.,Schoehn, G. (deposition date: 2016-06-25, release date: 2016-08-10, Last modification date: 2024-05-15)

Primary citation

Coscia, F.,Estrozi, L.F.,Hans, F.,Malet, H.,Noirclerc-Savoye, M.,Schoehn, G.,Petosa, C.
Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.
Sci Rep, 6:30909-30909, 2016

PubMed 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.

PubMed: 27485862
DOI: 10.1038/srep30909

Experimental method

ELECTRON MICROSCOPY (6.2 Å)