A perfect macromolecular structure would provide an all-atom description of the molecule, including not only the well-ordered polypeptide or polynucleotide framework but all other species: metals and other ions, cofactors, lipids, substrates and inhibitors. However, current structural data include no or very little information on elemental composition, leading to significant errors and omissions in atomic models. To address this issue, we are developing a method, Reconstructed Electron Energy Loss (REEL) analysis, to map elemental distribution within macromolecular complexes by bringing together well-established principles in analytical electron microscopy (EM) and biological cryogenic EM.
Atomic-resolution elemental mapping in the electron microscope is well established for dose-tolerant samples. Electron Energy Loss (EEL) techniques capture information from inelastic scattering events in the sample, and energy losses are characteristic of the element and chemical state of the scattering atom. These techniques require a high electron dose to achieve useable signal-to-noise ratio, severely limiting their application to biological samples.
Our approach combines the image processing tools of single-particle cryo-EM with EEL techniques, allowing us to add EEL signal in the 3D particle space, effectively dividing the dose required for sensitive elemental analysis between many images. We will apply our methodology to two important macromolecular complexes: the skeletal muscle ryanodine receptor and the mitochondrial F-type ATP synthase. We anticipate that such a method would be very impactful in understanding not only these complexes, but a wide variety of different biomolecules, including ion channels and other membrane proteins, complexes involved in transcription and translation, and metalloproteins, to name just a few. To make the technique broadly applicable in the life sciences, we aim to reach single-atom sensitivity and single-nanometer resolution in the context of this five-year project.
