Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins
Open accessPublished version of a preprint
Abstract
Significance: Structural studies of membrane proteins by cryogenic electron microscopy (cryo-EM) often require antibody fragments (Fabs) to facilitate particle alignments and achieve high resolution. While conformational nanobodies have been developed to lock specific states of many membrane proteins, they only add 15 kDa of mass to the complex. We developed a synthetic Fab (NabFab) that rigidly binds the conserved scaffold of nanobodies, providing a universally applicable fiducial for cryo-EM studies of protein–nanobody complexes. We demonstrate the concept by determining two high-resolution structures of membrane proteins bound to specific nanobodies and NabFab. As the structural epitope for NabFab can be incorporated into the scaffold of virtually any nanobody, this raises the prospect of facile structure determination of many nanobody–protein complexes.
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Cites
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Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter
Hariharan, P., Shi, Y., Katsube, S., Willibal, K., Burrows, N. D., Mitchell, P., Bakhtiiari, A., Stanfield, S., Pardon, E., Kaback, H. R., et al. (2024) · doi:10.7554/elife.92462.2
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- Authors
- Bloch, J. S., Mukherjee, S., Kowal, J., Filippova, E. V., Niederer, M., Pardon, E., Steyaert, J., Kossiakoff, A. A., & Locher, K. P.
- Affiliations
- ETH Zurich (CH)
- University of Chicago (US)
- Vrije Universiteit Brussel (BE)
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2021-11-15
- Volume
- 118
- Issue
- 47
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC8617411
- OpenAlex
- W3211372884
- Cited by
- 114
- References
- 3
- Citations by year
- 2026: 29 · 2025: 38 · 2024: 17 · 2023: 21 · 2022: 9
- FWCI
- 4.92
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- VUBIR (Vrije Universiteit Brussel) (submittedVersion)
- Funders
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_189111)
- Foundation for the National Institutes of Health (GM117372)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- nanobodies, fiducial markers, membrane proteins, high-resolution structure determination
- MeSH terms
- Animals, Camelids, New World, Camelus, Immunoglobulin Fab Fragments, Membrane Proteins, Microscopy, Electron, Models, Molecular, Protein Binding, Protein Conformation, Sequence Alignment, Cryoelectron Microscopy, Sequence Analysis, Protein, Single-Domain Antibodies
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