The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bactcomplex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5′ splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile—the branchsite adenosine—is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5′ss. Our structure suggests that Prp2 adenosine triphosphatase–mediated remodeling leads to conformational changes in Hsh155’s HEAT domain that liberate the first-step reactants for catalysis.
