Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

• Published in: Nature methods Vol. 18; no. 1; pp. 60 - 68
• Main Authors: Uchański, Tomasz, Masiulis, Simonas, Fischer, Baptiste, Kalichuk, Valentina, López-Sánchez, Uriel, Zarkadas, Eleftherios, Weckener, Miriam, Sente, Andrija, Ward, Philip, Wohlkönig, Alexandre, Zögg, Thomas, Remaut, Han, Naismith, James H., Nury, Hugues, Vranken, Wim, Aricescu, A. Radu, Pardon, Els, Steyaert, Jan
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-01-2021; Nature Publishing Group
• Subjects: 631/45; 631/61/338; Affinity; Antigens; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Cryoelectron Microscopy - methods; Electron microscopy; Grafting; Heterogeneity; Humans; Interfaces; Life Sciences; Lipids - chemistry; Membrane proteins; Membranes; Methods; Models, Molecular; Molecular Structure; Molecular weight; Multiprotein Complexes - chemistry; Nanobodies; Nanoparticles; Orientation; Particle size; Protein Conformation; Protein structure; Proteins; Proteomics; Receptors, GABA-A - chemistry; Scaffolds; Single Molecule Imaging - methods; Single-Cell Analysis - methods; Single-Domain Antibodies - chemistry; Structure; Toolkits; Yeasts; Belgium
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-020-01001-6

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
Megabodies, built by grafting nanobodies onto larger protein scaffolds, help alleviate problems of particle size and preferential orientation at the water–air interfaces during cryo-EM based structure determination experiments and are shown to be generalizable to soluble and membrane-bound proteins.