Eisosomes, stress-sensitive membrane compartments in yeast, are scaffolded by the BAR-domain proteins Pil1 and Lsp1. We isolated near-native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structure by helical reconstruction. From the same near-native eisosome samples, we performed mass spectroscopy to determine the protein content, and the abundance ratio of Pil1/Lsp1.