Bringing colour to cryo-EM: applying 3D reconstruction to electron energy loss spectral data for low-dose elemental mapping

Bonnie Murphy

Max Planck Institute of Biophysics

To build accurate atomic models and understand how a protein’s structure relates to its function, we would like our structures to accurately describe not only the polypeptide scaffold of a complex, but the full complement of associated metals and other ions, substrates and inhibitors, and lipids that interact with it. Unfortunately, interpreting experimental densities to full, accurate atomic models is often error-prone, because we have no way to experimentally map individual elements within our complexes. Several techniques exist for elemental mapping in the electron microscope, but these require very high doses that are generally incompatible with cryo-preserved biological samples. Using single-particle reconstruction (SPR), a high total dose can be spread across many (near-)identical copies of a given complex, such that signal-to-noise ratio can be built up from many images. We have applied SPR to images of protein complexes acquired in scanning transmission electron microscopy - electron energy loss spectroscopy (STEM-EELS) mode at fluences below 100 e/Å2. We estimate particle poses using higher-SNR reference images, and apply this pose information to sequentially reconstruct 3D maps across the electron energy loss spectrum. In this way, we generate three-dimensional reconstructions that reflect the distribution of abundant elements in the complex. I will discuss our established workflow, proof-of-principle results, and challenges and next steps required to reach single-atom sensitivity with this technique.

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