Intron removal requires assembly of the spliceosome on pre-mRNA and extensive remodelling to form the spliceosome’s catalytic centre. Here we report the cryo-electron microscopy structure of the yeast pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 snRNP associates with U4/U6.U5 tri-snRNP through U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The U2 snRNP 3’ region is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5’-splice site. The U6 ACAGAGA sequence forms a hairpin which weakly tethers the 5’-splice site. B complex proteins Prp38, Snu23, and Spp381 bind the Prp8 N-terminal domain and stabilise U6 ACAGAGA stem–pre-mRNA and Brr2–U4 snRNA interactions. These results provide important insights into the events leading to active site formation.