Three-dimensional Cryogenic Electron Microscopy Imaging of Two-dimensional Polymer Crystals*

We introduce a novel composite holey gold support that prevents cryo-crinkling and reduces beam-induced motion of two-dimensional specimens. The experiments are conducted on self-assembled crystalline polypeptoid nanosheets. Low-dose cryogenic electron microscopy micrographs are obtained from frozen hydrated crystalline nanosheets on the novel ultra-flat support at different tilting angles. A hybrid processing of crystallographic, tomography and single particle methods, developed for cryo-electron microscopy of biological macromolecules, is used to resolve the structure of crystals at atomic level. Our approach is robust and enable direct visualization of the spatial arrangement of atoms in backbones and side chains in the three-dimensional reconstructed crystalline polypeptoid nanosheet.