TY - JOUR
T1 - Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM
AU - Uchański, Tomasz
AU - Masiulis, Simonas
AU - Fischer, Baptiste
AU - Kalichuk, Valentina
AU - López-Sánchez, Uriel
AU - Zarkadas, Eleftherios
AU - Weckener, Miriam
AU - Sente, Andrija
AU - Ward, Philip
AU - Wohlkönig, Alexandre
AU - Zögg, Thomas
AU - Remaut, Han
AU - Naismith, James H.
AU - Nury, Hugues
AU - Vranken, Wim
AU - Aricescu, A. Radu
AU - Pardon, Els
AU - Steyaert, Jan
N1 - Funding Information:
We thank A.V. Shkumatov and R.K. Singh for support with SAXS experiments, and H. De Greve for providing the GFP+-expressing E. coli strain. We thank Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI), Instruct-ULTRA (EU H2020 grant no. 731005) and the Research Foundation—Flanders (FWO) for support with nanobody discovery and for funding the PhD training of T.U. We thank E. Beke for the technical assistance during megabody recloning. We thank Diamond Light Source, Harwell, UK, for access to crystallographic beamlines I03 and I24, and SAXS beamline B21. Cryo-EM studies of GABAA receptor were supported by the UK Medical Research Council grants no. MR/L009609/1 and no. MC_UP_1201/15 to A.R.A. We thank S. Chen, G. Cannone, G. Sharov and A. Yeates for support at the MRC-LMB EM facility; and J. Grimmett, T. Darling and T. Pratt for help with IT and high-performance computing. Cryo-EM studies of 5-HT3A receptor were supported by the ERC Starting grant no. 637733 Pentabrain, and the Fondation pour la Recherche Médicale grant no. SPF201809007073 to U.L.-S. We thank G. Schoehn and the IBS electron microscopy facility, supported by the Rhône-Alpes Region, the FRM, the FEDER and the GIS-IBISA. Cryo-EM studies of WbaP transferase were performed at Oxford Particle Imaging Centre founded by a Wellcome Trust JIF award (grant no. 060208/Z/00/Z) and supported by equipment grants from WT (grant no. 093305/Z/10/Z). We thank B. Qureshi for support with electron microscopy.
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85098856150&partnerID=8YFLogxK
U2 - 10.1038/s41592-020-01001-6
DO - 10.1038/s41592-020-01001-6
M3 - Article
C2 - 33408403
AN - SCOPUS:85098856150
SN - 1548-7091
VL - 18
SP - 60
EP - 68
JO - Nature Methods
JF - Nature Methods
IS - 1
ER -