Olivia Pfeil-Gardiner, Higor Vinícius Dias Rosa, Dietmar Riedel, Yu Seby Chen, Dominique Lörks, Pirmin Kükelhan, Martin Linck, Heiko Müller, Filip Van Petegem, Bonnie J. Murphy
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Elemental mapping in single-particle reconstructions by reconstructed electron energy-loss analysis
For macromolecular structures determined by cryogenic electron microscopy, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here we combine electron energy-loss spectroscopy with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, reconstructed electron energy-loss (REEL) analysis, allows a three-dimensional reconstruction of electron energy-loss spectroscopy data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localize abundant elements. We discuss the current limitations of the method and potential future developments. An approach combining electron energy-loss spectroscopy with image processing tools from single-particle cryo-electron microscopy enables elemental mapping in macromolecular complexes, paving the way for the accurate assignment of metals, ions, ligands and lipids.
期刊介绍:
Nature Methods is a monthly journal that focuses on publishing innovative methods and substantial enhancements to fundamental life sciences research techniques. Geared towards a diverse, interdisciplinary readership of researchers in academia and industry engaged in laboratory work, the journal offers new tools for research and emphasizes the immediate practical significance of the featured work. It publishes primary research papers and reviews recent technical and methodological advancements, with a particular interest in primary methods papers relevant to the biological and biomedical sciences. This includes methods rooted in chemistry with practical applications for studying biological problems.