Journal: Nat Methods / Year: 2021 Title: Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM. Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre ...Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre Wohlkönig / Thomas Zögg / Han Remaut / James H Naismith / Hugues Nury / Wim Vranken / A Radu Aricescu / Els Pardon / Jan Steyaert / 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 ...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.
Mass: 56165.656 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lama glama (llama), (gene. exp.) Helicobacter pylori (strain G27) (bacteria) Gene: Nb207, hopQ, HPG27_1120 / Strain: G27 / Production host: Escherichia coli (E. coli) / References: UniProt: B5Z8H1
Sequence details
Megabody Mb-Nb207-c7HopQ_G10 is a chimeric protein with circular permutation of HopQ: Residues 1-12: ...Megabody Mb-Nb207-c7HopQ_G10 is a chimeric protein with circular permutation of HopQ: Residues 1-12: a part of a β-strand A of the Nanobody fold. Residue 13: one amino acid linker. Residues 14-232: C-terminal part of HopQ (residues 228-446, UniProtKB B5Z8H1). Residues 233-400: N-terminal part of HopQ (residues 53-220, UniProtKB B5Z8H1). Residue 401: one amino acid linker. Residues 402-511: a part of the Nanobody fold. Residues 512-517: the His6 tag. Residues 518-521: the EPEA tag.
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.35 Å3/Da / Density % sol: 47.7 %
Crystal grow
Temperature: 293 K / Method: vapor diffusion / pH: 7.5 / Details: 0.1 M MgCl2, 0.1 M HEPES pH 7.5, 19% PEG 4000
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Data collection
Diffraction
Mean temperature: 100 K / Serial crystal experiment: N
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