Detection of Zn Atoms on Ferritin by Annular Dark-Field cryo-STEM

Abstract

Metal ions play essential roles in many aspects of biological chemistry, including oxygen transport, enzyme catalysis, and maintenance of biopolymer integrity. Cryo-electron microscopy is in principle sensitive to metal ions due to the stronger Coulomb potential relative to surrounding light elements, but conventional defocus phase contrast may not be the ideal approach to visualization. In tomography even small gold nanoparticles (<5 nm) are difficult to identify.We explore the alternative modality of scanning transmission EM (STEM), recently applied to cryo-microscopy and tomography. Annular dark-field STEM provides a quantitative image contrast based on atomic scattering, and has been exploited most effectively for mass measurements of macromolecules. Angular resolution of the scattering can provide further information to distinguish relatively heavy and light elements; in materials science this is known as Z contrast. We ask whether cryo-STEM can detect the presence and location of metal ions in protein complexes.Ferritin proteins forms a nearly spherical, highly symmetrical 3D shell structure by self-assembly of 24 polypeptide subunits. Ferritins bind iron as the primary physiological substrate, but also other metals. Zn binds to ferritin at precise ferroxidase sites within the structure. Taking advantage of these properties, we used cryo-STEM to identify Zn ions within the protein structure. Images were processed by single-particle alignment and averaging. Even in very small datasets, Zn stands out clearly at the predicted binding sites. The data provide an experimental measure of the signal contribution from single Zn atoms in cryo-STEM. This is key to understanding atomic detection of metals in 3D macromolecular and cellular contexts, as well as to use of synthetic metal tags as specific molecular labels.