Abstract: Membrane proteins present unique challenges in single-particle analysis (SPA) workflows, spanning the entire pipeline from sample purification and grid preparation to computational data processing. Their intrinsic structural heterogeneity, dependence on detergents or lipid environments, preferred particle orientation, and often low image contrast can complicate vitrification and imaging. In addition, conformational variability and compositional instability frequently limit efficient particle classification and high-resolution refinement. In this talk, I will discuss practical strategies to address these challenges, including optimization of biochemical conditions, grid preparation methods, and modern data-processing approaches designed to accommodate structural flexibility.
To illustrate these concepts, I will present a case study from our laboratory on determining the structure of the membrane proteins. These example will highlight real-world troubleshooting, from improving sample stability and mitigating orientation bias to extracting biologically meaningful conformational states from noisy datasets. Together, these perspectives aim to provide a realistic understanding of SPA workflows, the common obstacles encountered in membrane protein structural biology, and the practical strategies that enable successful structure determination.