Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, for particles smaller than 50kDa, single particle cryo-EM often fails due to the limited imaging contrast of individual micrographs. For dose-resilient samples often studied in the physical sciences, electron ptychography, a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM), has recently demonstrated resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied ptychographic data analysis to frozen hydrated single protein particles, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high speed of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan position, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong contrast enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Angstrom resolution. Ptychography is a promising tool to study the structure of smaller frozen hydrated protein particles, and, once combined with electron tomography tilt series, bears the potential to provide unique insight into the ultrastructure of vitrified biological tissue. ### Competing Interest Statement The authors have declared no competing interest.