Covalently constrained ‘Di-Gembodies’ enable parallel structure solutions by cryo-EM
Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
Authors
- Robert J.C. Gilbert (ORCID: https://orcid.org/0000-0001-9336-5604)
- Loïc Carrique (ORCID: https://orcid.org/0000-0001-5332-8593)
- M. Fairhead (ORCID: https://orcid.org/0000-0001-5361-3933)
- Dimitrios Mamalis (ORCID: https://orcid.org/0000-0002-4396-7495)
- Gangshun Yi (ORCID: https://orcid.org/0009-0004-6509-4893)
- Mingda Ye (ORCID: https://orcid.org/0000-0001-6324-4238)
- Huanyu Li (ORCID: https://orcid.org/0000-0002-5482-9936)
- Benjamin G. Davis (ORCID: https://orcid.org/0000-0002-5056-407X)
- F. von Delft (ORCID: https://orcid.org/0000-0003-0378-0017)
- David B. Sauer (ORCID: https://orcid.org/0000-0001-9291-4640)
- Katharina Duerr (ORCID: https://orcid.org/0000-0002-3245-4088)
- Peijun Zhang (ORCID: https://orcid.org/0000-0003-1803-691X)
Institutions
- Centre for Human Genetics (GB)
- Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
- University of Johannesburg (ZA)
- Northeast Catholic College (US)
- Discovery Centre (CA)
- Diamond Light Source (GB)
- University of Oxford (GB)
- Research Complex at Harwell (GB)
- Rosalind Franklin Institute (GB)
- e-Therapeutics (United Kingdom) (GB)
- Oxford Centre for Diabetes, Endocrinology and Metabolism (GB)
- Oxford BioMedica (United Kingdom) (GB)
- Science Oxford (GB)
Publication Details
- Journal
- Nature Chemical Biology
- Published
- 2025-08-15
- DOI
- https://doi.org/10.1038/s41589-025-01972-7
- Citations
- 8
- Primary Topic
- Advanced Electron Microscopy Techniques and Applications
- Type
- article
- Field-Weighted Citation Impact
- 20.39