This study presents a method for achieving sub-nanometer resolution 3D reconstructions of proteins using low-dose cryo-electron ptychography. The researchers applied ptychographic data analysis to frozen hydrated single protein particles, achieving sub-nanometer resolution. They used an aberration-corrected, convergent electron beam to collect 4D-STEM data, enabling the reconstruction of the structure of apoferritin at up to 5.8 Å resolution. Ptychography is a promising tool for studying smaller frozen hydrated protein particles and, when combined with electron tomography tilt series, can provide unique insights into the ultrastructure of vitrified biological tissue. Cryo-EM has revolutionized life sciences and pharmaceutical research, but faces challenges with small proteins due to limited imaging contrast. Electron ptychography, a coherent diffractive imaging technique using 4D-STEM, has shown promise in achieving high resolution for thin specimens. The study demonstrates that low-dose cryo-EM with 4D-STEM can achieve sub-nanometer resolution for protein structures, using a defocused beam and ptychographic data processing. The method allows for the reconstruction of protein structures from fewer particles than typically imaged in cryo-EM. The study applied 4D-STEM to apoferritin, phi92 bacteriophage, and TMV, achieving resolutions of 5.8 Å, 8.4 Å, and 6.4 Å, respectively. The results show that 4D-STEM ptychography can provide high-resolution structural information of biological specimens. The method involves the use of a pixelated detector and advanced data processing algorithms to handle the large datasets generated by 4D-STEM. The study also highlights the importance of optimizing data collection parameters to minimize the signal-to-noise ratio and improve resolution. The results demonstrate that 4D-STEM ptychography can achieve high-resolution structural information of biological specimens, with potential applications in the study of cellular structures and biological tissues. The method is promising for structural biology and has the potential to provide unique insights into the ultrastructure of vitrified biological tissue. The study also highlights the need for further optimization of data collection and processing parameters to improve resolution and reduce the signal-to-noise ratio.This study presents a method for achieving sub-nanometer resolution 3D reconstructions of proteins using low-dose cryo-electron ptychography. The researchers applied ptychographic data analysis to frozen hydrated single protein particles, achieving sub-nanometer resolution. They used an aberration-corrected, convergent electron beam to collect 4D-STEM data, enabling the reconstruction of the structure of apoferritin at up to 5.8 Å resolution. Ptychography is a promising tool for studying smaller frozen hydrated protein particles and, when combined with electron tomography tilt series, can provide unique insights into the ultrastructure of vitrified biological tissue. Cryo-EM has revolutionized life sciences and pharmaceutical research, but faces challenges with small proteins due to limited imaging contrast. Electron ptychography, a coherent diffractive imaging technique using 4D-STEM, has shown promise in achieving high resolution for thin specimens. The study demonstrates that low-dose cryo-EM with 4D-STEM can achieve sub-nanometer resolution for protein structures, using a defocused beam and ptychographic data processing. The method allows for the reconstruction of protein structures from fewer particles than typically imaged in cryo-EM. The study applied 4D-STEM to apoferritin, phi92 bacteriophage, and TMV, achieving resolutions of 5.8 Å, 8.4 Å, and 6.4 Å, respectively. The results show that 4D-STEM ptychography can provide high-resolution structural information of biological specimens. The method involves the use of a pixelated detector and advanced data processing algorithms to handle the large datasets generated by 4D-STEM. The study also highlights the importance of optimizing data collection parameters to minimize the signal-to-noise ratio and improve resolution. The results demonstrate that 4D-STEM ptychography can achieve high-resolution structural information of biological specimens, with potential applications in the study of cellular structures and biological tissues. The method is promising for structural biology and has the potential to provide unique insights into the ultrastructure of vitrified biological tissue. The study also highlights the need for further optimization of data collection and processing parameters to improve resolution and reduce the signal-to-noise ratio.