Microcrystal Electron Diffraction-Guided Discovery of Fungal Metabolites
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1.
California Institute of Technology
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2.
University of California, Los Angeles
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3.
The University of Texas Southwestern Medical Center
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4.
The University of Texas Health Science Center at Houston
Abstract
Nature remains a vast repository of complex and functional metabolites whose structural characterization continues to drive innovations in pharmaceuticals, agrochemicals, and materials science. The cryogenic electron microscopy (cryoEM) method, microcrystal electron diffraction (microED, a 3D ED technique), has emerged as a powerful tool to structurally characterize small molecules. Despite this emerging role in structural chemistry, the cost and throughput of microED have limited its application in the discovery of natural products (NPs). While recent advances in sample preparation (e.g., arrayED) have provided a conceptual framework to address these challenges, they have remained unproven. Herein, we report the arrayED-driven discovery of a structurally unprecedented family of NPs (zopalides A-E), a muurolane-type sesquiterpene glycoside (rhytidoside A), aspergillicin analogs (aspergillicins H and I), and four crystal structures of previously reported fungal metabolites. Lastly, this the first time that the absolute stereo configuration of newly discovered NPs has been determined directly by microED alone without other methods using a small amount of sample.
Copyright and License
© 2025 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY-NC-ND 4.0 .
Supplemental Material
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Detailed experimental protocols on sample preparation of fungal culture, extraction procedure, 96-well fractionation scheme, and compound isolation procedure; supporting data for arrayED screening, structural models, and NMR characterization; and preliminary solutions and fully refined structural solutions for identified crystalline natural products (PDF): ja5c01466_si_001.pdf
Data Availability
The raw microED data for this study are available on Zenodo. The autoprocessing Python script utilized in this study is available at GitHub.
CCDC 2417602–2417612 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via the joint Cambridge Crystallographic Data Centre (CCDC) and Fachinformationszentrum Karlsruhe Access Structures service.
Acknowledgement
We thank Dr. Songye Chen (Caltech) and the Caltech cryo-EM facility for materials and advice, Dr. Scott Virgil (Caltech) for expertise with instrumentation, Donald W. Crocker for helpful discussions, the Resnick High Performance Computing Cluster (Caltech) for computational resources, and Dr. Michael Takase (Caltech) for assistance with X-ray crystallography. I.H.R. is supported by the NSF Graduate Research Fellowship under grant 2139433. This research was funded by NIH NCCIH 1 R01AT011990 (H.M.N. and Y.T.), the HHMI Emerging Pathogens Initiative, the Packard Foundation (H.M.N.), and the Pew Charitable Trust (H.M.N.).
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Additional details
- Howard Hughes Medical Institute
- Emerging Pathogens Initiative -
- National Center for Complementary and Integrative Health
- R01AT011990
- David and Lucile Packard Foundation
- Pew Charitable Trusts
- Resnick Sustainability Institute
- National Science Foundation
- Graduate Research Fellowship 2139433
- Caltech groups
- Division of Chemistry and Chemical Engineering (CCE)
- Publication Status
- In Press