Extended Data Fig. 6: Structural changes observed after addition of the 5’ss oligo alone or the 5’ss oligo plus ATPγS.

a, Fit of a single 5’ss oligo to the pre-B^5’ss EM density. b, The movement of the PRP8^RT/En domain toward the PRP8^NTD upon addition of the 5’ss oligo leads to concomitant movements of other tri-snRNP components that interact with PRP8^RT/En. Comparison of the molecular architecture of BRR2, PRP8, SAD1 and the U4 Sm core domain in pre-B (left) and pre-B^5’ss complexes (middle). Right, overlay of the corresponding surface representations in pre-B (grey) versus pre-B^5’ss (various colors), with arrows indicating the concomitant movements of the indicated domains with PRP8^RT/En. These movements do not involve any major structural changes such as translocation of BRR2 to its activation position. c,d, Comparison of the molecular architecture of PRP4 kinase, SF3B3, PRP6^HAT, SNU66 and the U4 Sm core domain in pre-B (panel c, left) and pre-B^5’ss complexes (panel d, left). The boxed regions are expanded and rotated 90° in the corresponding panels shown at the right. Aligned via the PRP8^RT/En domain. In pre-B^5’ss, PRP4K and SF3B3^WD40B are located further apart, and PRP4K is more closely associated with PRP6^HAT. Moreover, the U4 Sm core has moved away from PRP8^RH in pre-B^5’ss (as indicated by the arrow in panel c). e, Overlay of pre-B and pre-B^5’ss showing differences in the position of PRP4K. f, In pre-B^5’ss, BRR2^PWI is detached from SAD1. A comparison of the molecular architecture of SAD1, BRR2^CC and BRR2^PWI in pre-B (panel f, left) and pre-B^5’ss complexes (panel f, middle). An overlay of the structures is shown in panel f (right); alignment on PRP8^NTD. g, Interactions of the 5’ss-containing RNA oligonucleotide (5’ss oligo) with U5 snRNA loop 1 nucleotides, and residues of PRP8 and DIM1 in pre-B^{5’ss+ATPγS}. h, Fit of the nucleotides and protein side chains shown in panel g into the pre-B^{5’ss+ATPγS} EM density. i, Rearrangements in the NOP and adjacent coiled-coil domains of PRP31 (aa 52-331) and repositioning of the PRP6 HAT domain and its more N-terminal phosphorylated region upon addition of ATP to pre-B^5’ss complexes. The coiled/coil (CC) domains of PRP31 rotate by ca 45° relative to the PRP31 NOP domain after addition of ATP. A comparison of the molecular architecture of the indicated proteins/protein domains in pre-B^5’ss (left), pre-B^{5’ss+ATPγS} (middle) and B-like complexes (right). Serine and threonine residues of PRP6 that could be modelled previously^18,19 and that were previously shown to be phosphorylated by PRP4K²⁶ are indicated by green stars. The region of PRP31 that is known to be phosphorylated could not be modelled. j, ATP addition to pre-B^5’ss leads to the ca 180° rotation of PRP8^RH, which is coordinated with the repositioning of BRR2/PRP8^Jab1, PRP6 and SNU66, among others. The β-hairpin loop of PRP8^RH is indicated in yellow and by an arrowhead. The SNU66 domain comprised of aa 252-358 is structured first in pre-B^{5’ss+ATPγS} where it is docked to the β-hairpin loop region of the repositioned PRP8^RH domain (compare left and middle panels). The C-terminal SNU66^630-774 domain, which binds across SNRP27K and the U4 Sm core in pre-B^5’ss, is also repositioned in pre-B^{5’ss+ATPγS}.