CryoFAST: A Fab-Aided Cryo-EM Workflow for High-Resolution Structure Determination of Sub-50-kDa Protein Targets
- 1Cryo-EM Platform, VIVA Biotech, Shanghai, China
Abstract
Single-particle cryo-electron microscopy (cryo-EM) plays an increasingly important role in structure-based drug discovery (SBDD) by revealing target protein-ligand interactions at the molecular level, but its application has largely focused on large- and medium-sized protein complexes. Many therapeutically important targets are smaller than 50 kDa and remain highly challenging for cryo-EM analysis because of low signal-to-noise ratios and difficulties in particle alignment. Here, we describe cryoFAST, an integrated workflow that couples ligand-compatible Fab discovery and structure-guided computational maturation with biochemical triage, fiducial-complex assembly, and single-particle cryo-EM. Through phage-display screening and/or AI-guided design, we developed high-affinity Fab binders of approximately 50 kDa that stabilize the target without disrupting ligand-target interactions and then assembled Fab-target complexes for cryo-EM analysis. Using the therapeutically relevant extracellular domain (~20 kDa) of interleukin-4 receptor (IL-4R) as a proof of concept, we determined its cryo-EM structure at 2.74 Angstrom resolution and established a framework for elucidating ligand-binding modes. This workflow provides a practical, efficient, and potentially broadly applicable route to the structural characterization of small protein targets that are otherwise challenging to study. By extending the applicability of cryo-EM to high-value but technically challenging small targets, cryoFAST may open new opportunities for structure-based drug discovery.
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BibTeX
@misc{li2026cryofast,
title = {{CryoFAST: A Fab-Aided Cryo-EM Workflow for High-Resolution Structure Determination of Sub-50-kDa Protein Targets}},
author = {X. Li and S. Deng and W. Li and Y. Qian and Y. Li and J. Du and L. Li and D. Qian},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.07.757472},
url = {https://www.biorxiv.org/content/10.64898/2026.10.07.757472v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - CryoFAST: A Fab-Aided Cryo-EM Workflow for High-Resolution Structure Determination of Sub-50-kDa Protein Targets
AU - X. Li
AU - S. Deng
AU - W. Li
AU - Y. Qian
AU - Y. Li
AU - J. Du
AU - L. Li
AU - D. Qian
PY - 2026
DA - 2026/10/07
PB - bioRxiv
DO - 10.64898/2026.10.07.757472
UR - https://www.biorxiv.org/content/10.64898/2026.10.07.757472v1
AB - Single-particle cryo-electron microscopy (cryo-EM) plays an increasingly important role in structure-based drug discovery (SBDD) by revealing target protein-ligand interactions at the molecular level, but its application has largely focused on large- and medium-sized protein complexes. Many therapeutically important targets are smaller than 50 kDa and remain highly challenging for cryo-EM analysis because of low signal-to-noise ratios and difficulties in particle alignment. Here, we describe cryoFAST, an integrated workflow that couples ligand-compatible Fab discovery and structure-guided computational maturation with biochemical triage, fiducial-complex assembly, and single-particle cryo-EM. Through phage-display screening and/or AI-guided design, we developed high-affinity Fab binders of approximately 50 kDa that stabilize the target without disrupting ligand-target interactions and then assembled Fab-target complexes for cryo-EM analysis. Using the therapeutically relevant extracellular domain (~20 kDa) of interleukin-4 receptor (IL-4R) as a proof of concept, we determined its cryo-EM structure at 2.74 Angstrom resolution and established a framework for elucidating ligand-binding modes. This workflow provides a practical, efficient, and potentially broadly applicable route to the structural characterization of small protein targets that are otherwise challenging to study. By extending the applicability of cryo-EM to high-value but technically challenging small targets, cryoFAST may open new opportunities for structure-based drug discovery.
ER -