In situ structure determination of conformationally flexible targets using nextPYP.

Single-particle cryoelectron tomography (SP-CET) is an imaging technique capable of determining the structure of proteins in their cellular environment at high-resolution. nextPYP is a web-based application designed to streamline the SP-CET structure determination process and facilitate the analysis of conformational variability. Here we explain how to use nextPYP-based methods to determine the structure and study the conformational heterogeneity of proteins using SP-CET. We provide a step-by-step guide to convert raw tilt-series into three-dimensional structures, following a workflow that includes movie-frame alignment, tilt-series alignment, contrast transfer function estimation, tomogram reconstruction, particle picking, high-resolution refinement and three-dimensional classification. The advantages of nextPYP are its ease-of-use and effectiveness at extracting high-resolution information, which, combined with its storage and compute efficiency, shortens time-to-structure from months to days. The complete procedure, including interactive data analysis and visualization, is fully integrated within the application, with no need for external software. Starting from raw tilt-series data, users will be able to determine a near-atomic resolution structure of human immunodeficiency virus 1 Gag protein, eight translational states of Escherichia coli 70S ribosomes and a structure of human 80S ribosomes from plasma-focused ion beam milled HeLa cells. This Protocol is intended as a resource for those who are new to SP-CET as well as more experienced users that want to streamline the process of in situ structure determination and heterogeneity analysis using nextPYP. The procedure requires 18 h to complete.