gamma-Secretase belongs to an atypical class of aspartic proteases that hydrolyzes peptide bonds within the transmembrane domain of substrates, including amyloid-beta precursor protein and Notch. gamma-Secretase is comprised of presenilin, nicastrin, APH-1, and PEN-2 which form a large multimeric membrane protein complex, the three-dimensional structure of which is unknown. To gain insight into the structure of this complex enzyme, we purified functional gamma-secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of gamma-secretase at a resolution of 48 angstrom. The structure occupies a volume of 560 x 320 x 240 angstrom and resembles a flat heart comprised of two oppositely faced, dimpled domains. A low density space containing multiple pores resides between the domains. Some of the dimples in the putative transmembrane region may house the catalytic site. The large dimensions are consistent with the observation that gamma-secretase activity resides within a high molecular weight complex. (c) 2006 Elsevier Inc. All rights reserved.