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

• Published in: Nature communications 2024-09, Vol.15 (1), p.8062-10, Article 8062
• Main Authors: Küçükoğlu, Berk, Mohammed, Inayathulla, Guerrero-Ferreira, Ricardo C., Ribet, Stephanie M., Varnavides, Georgios, Leidl, Max Leo, Lau, Kelvin, Nazarov, Sergey, Myasnikov, Alexander, Kube, Massimo, Radecke, Julika, Sachse, Carsten, Müller-Caspary, Knut, Ophus, Colin, Stahlberg, Henning
• Format: Article
• Language: English
• Subjects: 101/28; 147/137; 631/45; 631/535/1258/1259; 631/57; 639/624/1107/328/2082; Apoferritin; Apoferritins - chemistry; Apoferritins - ultrastructure; Bacteriorhodopsin; Cryoelectron Microscopy - methods; Data analysis; Data processing; Diffraction patterns; Electron beams; Electron diffraction; Electrons; Humanities and Social Sciences; Image Processing, Computer-Assisted - methods; Image reconstruction; Imaging techniques; Imaging, Three-Dimensional - methods; Micrography; Microscopy, Electron, Scanning Transmission - methods; multidisciplinary; Photomicrographs; Physical sciences; Protein structure; Proteins; Proteins - chemistry; Proteins - ultrastructure; Room temperature; Scanning electron microscopy; Scanning transmission electron microscopy; Science; Science (multidisciplinary); Sheaths; Signal to noise ratio; Structural analysis; Tobacco; Transmission electron microscopy
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-52403-5

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. 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 excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we apply 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employ low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allows us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples are iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 Å resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions. 4D-scanning transmission electron microscopy uses diffractive imaging for structural studies. Here, authors study single particle cryo-EM protein samples at up to 5.8 Å resolution, using 4D-STEM and ptychography data processing.