DETECTION OF ISOLATED METAL ATOMS ON FERRITIN BY CRYO-SCANNING TRANSMISSION ELECTRON MICROSCOPY

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 sensitive to metal ions because of their strong Coulomb potential relative to surrounding light elements. However the conventional mode of defocus phase contrast in wide-field transmission electron microscopy (TEM) is not well suited to visualization of single atoms. In practice even small gold nanoparticles (<5 nm) can be 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. Its traditional role in biological microscopy has been for mass measurements of macromolecules. We ask whether cryo-STEM can detect the presence and location of metal ions in protein complexes. As a test case we study Zn bound to ferritin. Ferritin proteins form a nearly spherical hollow shell structure by self-assembly of 24 polypeptide subunits. This assembly stores iron in mineralized form within the central cavity. Zn binds tightly to ferritin at precise ferroxidase sites where the iron undergoes oxidation en route to the center. Cryo-STEM images of Zn-loaded ferritin were processed by single-particle alignment and averaging. Even in very small datasets, the Zn stands out clearly at the predicted binding sites. Comparison with the raw images confirms detection of isolated metal 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.