Fast event-based electron counting for small-molecule structure determination by MicroED
Creators
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1.
University of Southern California
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2.
California Institute of Technology
Abstract
Electron counting helped realize the resolution revolution in single-particle cryoEM and is now accelerating the determination of MicroED structures. Its advantages are best demonstrated by new direct electron detectors capable of fast (kilohertz) event-based electron counting (EBEC). This strategy minimizes the inaccuracies introduced by coincidence loss (CL) and promises rapid determination of accurate structures. We used the Direct Electron Apollo camera to leverage EBEC technology for MicroED data collection. Given its ability to count single electrons, the Apollo collects high-quality MicroED data from organic small-molecule crystals illuminated with incident electron beam flux densities as low as 0.01–0.045 e−/Å2/s. Under even the lowest flux density (0.01 e−/Å2/s) condition, fast EBEC data produced ab initio structures of a salen ligand (268 Da) and biotin (244 Da). Each structure was determined from a 100° wedge of data collected from a single crystal in as few as 50 s, with a delivered fluence of only ∼0.5 e−/Å2. Fast EBEC data collected with a fluence of 2.25 or 3.33 e−/Å2 also facilitated a 1.5 Å structure of thiostrepton (1665 Da). While refinement of these structures appeared unaffected by CL, a CL adjustment applied to EBEC data further improved the distribution of intensities measured from the salen ligand and biotin crystals. However, CL adjustment only marginally improved the refinement of their corresponding structures, signaling the already high counting accuracy of detectors with counting rates in the kilohertz range. Overall, by delivering low-dose structure-worthy data, fast EBEC collection strategies open new possibilities for high-throughput MicroED.
Copyright and License
Published under a CC BY 4.0 licence.
Acknowledgement
We thank Drs Michael Sawaya (UCLA) and Ambarneil Saha (LBNL) for their input and helpful discussions. We thank Isabel Hernandez Rodriguez (Caltech) for her technical input on electron diffraction automation. We also thank Dr Benjamin Bammes (DE) for his technical guidance on the Direct Electron Apollo camera and helpful discussions on coincidence loss estimation. JAR is supported as a Packard Fellow. This work is also funded by the HHMI Emerging Pathogens Initiative. Structures determined from Apollo data of the salen ligand and biotin are deposited under CCDC deposition numbers 2370186 (salen ligand) and 2370185 (biotin), respectively, and a structure of thiostrepton is deposited under PDB accession code 9cq0. Diffraction data are available under Zenodo entries 13690162, 13713098, 13716076, and 13716389. Author contributions: NV, HMN, and JAR designed and guided experiments; NV and JAR prepared and characterized samples; NV, SQ, LSR, LSM, DC, and JAR measured and analyzed diffraction data; NV, SQ, and LSR determined and refined the structures. All authors helped write and provided critical feedback on the article.
Funding
Funding for this research was provided by: U.S. Department of Energy (grant No. DE-FC02-02ER63421); National Institutes of Health, National Institute of General Medical Sciences (grant No. R35 GM128867); Howard Hughes Medical Institute.
Supplemental Material
Crystal structure: contains datablocks a, a_1, global. DOI: https://doi.org/10.1107/S2053229624012300/yp3239sup1.cif
Scripts, tables and figures. DOI: https://doi.org/10.1107/S2053229624012300/yp3239sup2.pdf
Files
yp3239.pdf
Additional details
Identifiers
- PMID
- 39982366
- PMCID
- PMC11881165
Funding
- United States Department of Energy
- DE-FC02-02ER63421
- National Institute of General Medical Sciences
- R35 GM128867
- Howard Hughes Medical Institute
Dates
- Submitted
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2024-08-19
- Accepted
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2024-12-20
Caltech Custom Metadata
- Caltech groups
- Division of Chemistry and Chemical Engineering (CCE)
- Publication Status
- Published