This study demonstrates the application of low-dose cryo-electron ptychography to achieve sub-nanometer resolution 3D reconstructions of frozen hydrated protein particles. The technique, which combines 4D scanning transmission electron microscopy (4D-STEM) with electron ptychography, overcomes the limitations of conventional cryo-electron microscopy (cryo-EM) for small particles (≤50 kDa) due to low signal-to-noise ratios. By using an aberration-corrected, convergent electron beam and a fast hybrid pixel detector, the researchers collected large datasets of electron diffraction patterns with substantial overlaps between adjacent scan positions. These patterns were then iteratively reconstructed to obtain scattering potentials, which were used to reconstruct the protein structures. The method successfully achieved high-resolution reconstructions of apoferritin, bacteriophage Phi92, and tobacco mosaic virus (TMV), with resolutions of 5.8 Å, 8.4 Å, and 6.4 Å, respectively. The study highlights the potential of cryo-electron ptychography for studying the ultrastructure of vitrified biological tissues and smaller frozen hydrated protein particles.This study demonstrates the application of low-dose cryo-electron ptychography to achieve sub-nanometer resolution 3D reconstructions of frozen hydrated protein particles. The technique, which combines 4D scanning transmission electron microscopy (4D-STEM) with electron ptychography, overcomes the limitations of conventional cryo-electron microscopy (cryo-EM) for small particles (≤50 kDa) due to low signal-to-noise ratios. By using an aberration-corrected, convergent electron beam and a fast hybrid pixel detector, the researchers collected large datasets of electron diffraction patterns with substantial overlaps between adjacent scan positions. These patterns were then iteratively reconstructed to obtain scattering potentials, which were used to reconstruct the protein structures. The method successfully achieved high-resolution reconstructions of apoferritin, bacteriophage Phi92, and tobacco mosaic virus (TMV), with resolutions of 5.8 Å, 8.4 Å, and 6.4 Å, respectively. The study highlights the potential of cryo-electron ptychography for studying the ultrastructure of vitrified biological tissues and smaller frozen hydrated protein particles.