nextPYP: a comprehensive and scalable platform for characterizing protein variability in situ using single-particle cryo-ET

Single-particle cryo-electron tomography (SP-CET) is an emerging technique capable of determining the structure of proteins imaged within the native context of cells at molecular resolution. While high-throughput techniques for sample preparation and tilt-series acquisition are beginning to provide sufficient data to allow structural studies of proteins at physiological concentrations, the complex data analysis pipeline and the demanding storage and computational requirements pose major barriers for the development and broader adoption of this technology. Here, we present a scalable, end-to-end framework for SP-CET data analysis –from on-the-fly pre-processing of tilt-series to high-resolution refinement and classification– that allows efficient analysis and visualization of datasets with hundreds of tilt-series and hundreds of thousands of particles. We validate our approach using in-vitro and cellular datasets, demonstrating its effectiveness at achieving high-resolution and revealing conformational heterogeneity in-situ. The framework is made available through an intuitive and easy-to-use computer application, nextPYP (https://nextpyp.app).

 

Nature Methods, 2023.