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

DOI: 10.1038/s41592-020-01001-6

Authors: Tomasz Uchański (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Simonas Masiulis (MRC Laboratory of Molecular Biology) , Baptiste Fischer (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Valentina Kalichuk (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Uriel López-Sánchez (CNRS, Université Grenoble Alpes, CEA) , Eleftherios Zarkadas (CNRS, Université Grenoble Alpes, CEA) , Miriam Weckener (Rosalind Franklin Institute) , Andrija Sente (MRC Laboratory of Molecular Biology) , Philip Ward (Wellcome Centre for Human Genetics, University of Oxford) , Alexandre Wohlkonig (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Thomas Zogg (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Han Remaut (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , James Naismith (Rosalind Franklin Institute; Wellcome Centre for Human Genetics, University of Oxford) , Hugues Nury (CNRS, Université Grenoble Alpes, CEA) , Wim Vranken (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , A. Radu Aricescu (MRC Laboratory of Molecular Biology; Wellcome Centre for Human Genetics, University of Oxford) , Els Pardon (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB) , Jan Steyaert (Vrije Universiteit Brussel, VUB; VIB-VUB Center for Structural Biology, VIB)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Methods , VOL 18 , PAGES 60 - 68

State: Published (Approved)
Published: January 2021

Abstract: 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.

Subject Areas: Biology and Bio-materials, Technique Development


Instruments: B21-High Throughput SAXS , I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 13/01/2021 08:54

Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Scattering Macromolecular Crystallography (MX) Small Angle X-ray Scattering (SAXS)