Atomic-resolution cryo-electron ptychography for structural biology

Objective

This project aims to establish an automated cryo-electron ptychography (cryo-EP) workflow to achieve atomic-resolution (better than 4 Å) structural determination of biological macromolecules, including those under 50 kDa, which remain beyond the reach of conventional cryo-electron microscopy (cryo-EM). Cryo-EP offers superior contrast transfer and defocus tolerance compared to mainstream cryo-EM and cryo-integrated differential phase contrast (cryo-iDPC), yet its application in structural biology is hindered by challenges such as low-dose data acquisition, probe position inaccuracies, motion blur, and lack of integrated software. To address these limitations, we propose a multidisciplinary approach combining optimized electron-optical parameters, dose-fractionated acquisition, multi-convergence semi-angle (multi-CSA) imaging, and robust computational reconstruction algorithms. The project will develop an automated, open-source software pipeline for data acquisition and processing, leveraging state-of-the-art cryo-electron microscopes (e.g., Titan Krios G4, BIOTOOL system operating at liquid helium temperature), high-speed detectors (e.g., Dectris Arina, TimePix) and high-performance computing resources. Validation will be performed on benchmark samples (e.g., TMV, apoferritin) and extended to challenging targets such as hemoglobin (60 kDa) and fetuin A (40 kDa). By overcoming current technical barriers, this project will enable high-resolution structural insights into previously inaccessible biomolecules, with broad implications for drug design, biotechnology, and fundamental biology. The outcomes will be disseminated through open-access publications, software releases, and public engagement, ensuring wide adoption and sustainability within the structural biology community.