Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM
| Autor: | Alexandre Wohlkonig, Hugues Nury, Baptiste Fischer, Tomasz Uchański, Eleftherios Zarkadas, Philip N. Ward, Thomas Zögg, Wim F. Vranken, Uriel López-Sánchez, Han Remaut, Jan Steyaert, S. Masiulis, A. Radu Aricescu, Valentina Kalichuk, Els Pardon, Andrija Sente, Miriam Weckener, James H. Naismith |
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| Přispěvatelé: | Department of Bio-engineering Sciences, Structural Biology Brussels, Basic (bio-) Medical Sciences, Chemistry, Informatics and Applied Informatics, VIB-VUB Center for Structural Biology [Bruxelles], VIB [Belgium] |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Scaffold protein
Models Molecular Materials science Cryo-electron microscopy Protein Conformation [SDV]Life Sciences [q-bio] Immunoglobulin domain Yeast display Biochemistry Article 03 medical and health sciences Protein structure Humans Molecular Biology ComputingMilieux_MISCELLANEOUS 030304 developmental biology 0303 health sciences Molecular Structure Resolution (electron density) Cryoelectron Microscopy Cell Biology Single-Domain Antibodies Receptors GABA-A Lipids Single Molecule Imaging Structural biology Membrane protein Multiprotein Complexes Biophysics Single-Cell Analysis Biotechnology |
| Zdroj: | Nat Methods Nature Methods Nature Methods, Nature Publishing Group, 2021, 18 (1), pp.60-68. ⟨10.1038/s41592-020-01001-6⟩ |
| ISSN: | 1548-7091 1548-7105 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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