Development of Reconstructed Electron Energy Loss techniques for Elemental Mappi...
Development of Reconstructed Electron Energy Loss techniques for Elemental Mapping in macromolecular structures
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,...
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, I propose to develop a method, Reconstructed Electron Energy Loss - Elemental Mapping (REEL-EM), that will 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 novel 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. Preliminary work in my research group confirms that our proposed approach is valid - we are able to generate maps of specific elements in the 3D particle space. I propose to extend this early work to achieve single-atom detection at 1-nm spatial resolution in the course of this five-year project. Our work will characterise and optimise all aspects of data collection and processing for REEL-EM. We will apply our methodology to two important macromolecular complexes: the skeletal muscle ryanodine receptor and the mitochondrial F-type ATP synthase.ver más
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