j.str.2014.09.005 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). SUMMARY Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the cor-rect topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purifica-tion, and reconstitution required in classical mem-brane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryoto-mography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distri-bution and interprotein interactions on the mem-brane. MPEEVs have many applications beyond structural/functional investigations, such as facili-tating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as bio-markers, and possibly therapeutics. INTRODUCTION