Published January 26, 2025 | Submitted v2
Working Paper Open

Microcrystal electron diffraction-guided discovery of fungal metabolites

  • 1. ROR icon California Institute of Technology
  • 2. ROR icon University of California, Los Angeles
  • 3. ROR icon The University of Texas Southwestern Medical Center
  • 4. ROR icon 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 (zopalide A-E), a muurolane-type sesquiterpene glycoside (rhytidoside A), aspergillicin analogs (aspergillicin H and aspergillicin I), and four crystal structures of previously reported fungal metabolites. We provide the first examples of absolute stereochemistry determination via microED for newly annotat-ed NPs.

Copyright and License

The content is available under CC BY NC ND 4.0.

Funding

This research was funded by NIH NCCIH 1 R01AT011990 (H.M.N. and Y.T.), HHMI Emerging Pathogen Initiative, Packard Foundation (H.M.N.), and Pew Charitable Trust (H.M.N.).

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. 

Contributions

DAD and HMN conceptualized the workflow and methodology. DAD collected data and carried out the workflow. LW and YZ conducted the culturing, isolation experimentation, and NMR characterization. CW and BPT conducted the bioassay
experimentation. IHR and MJT screened wells and collected data. DAD, JEB, and KKJ carried out data processing and structural solution. LSM carried out dynamical refinement of compounds 1 and 6. KKJ carried out dynamical refinement of compound 5. GFB isolated and identified the fungal strains utilized in this study. HMN, YT, and BPT supervised experimentation. DAD and HMN wrote the original manuscript draft and HMN, KKJ, LW, CW, YT, and BPT edited the manuscript.

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 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax + 44 1223 336033.

Files

microcrystal-electron-diffraction-guided-discovery-of-fungal-metabolites.pdf

Additional details

Created:
March 3, 2025
Modified:
March 3, 2025