Extended Data Fig. 3: Cryo-EM and image-processing of the cross-exon B-like complex.

a, RNA composition of the purified B-like dimers. B-like complexes were affinity-purified and RNA from the fastest-sedimenting gradient peak was isolated, separated on a NuPAGE gel, and visualized by staining with SyBr gold. For gel source data, see Supplementary Fig. 1. The nucleotide (nts) lengths of the snRNAs and MINX exon RNA are indicated on the right. The RNA composition was analysed from four independent B-like complex purifications with similar results. U1 snRNA, which due to the presence of an excess of the 5’ss oligo should no longer be base paired to the 5’ss of the MINX exon RNA (see panel I) is still present, together with U2, U4, U5 and U6 snRNA, and the MINX exon RNA. It is very likely that, under our low salt purification conditions, U1 snRNP remains bound via protein-protein interactions presumably in the vicinity of the MINX 5’ss (i.e., in the upper poorly-resolved, globular EM density). Substantial amounts of U1 are also observed in all other complexes incubated in the presence of an excess of the 5’ss oligo (see below). b, Representative cryo-EM 2D class averages of the CE B-like dimers. c, Cryo-EM computation sorting scheme for CE B-like complexes. All major image-processing steps are depicted. The tri-snRNP core is comprised of all tri-snRNP components excluding the U4 Sm core, U5 Sm core and BRR2. The U4/U6 part consists of the U4/U6 stems I and II and associated proteins. d, Local resolution estimation of the tri-snRNP core region of the B-like complex. e, Orientation distribution plot for the particles contributing to the reconstruction of the tri-snRNP core region. f, Fourier shell correlation (FSC) values for the listed parts of the B-like complex, indicate a resolution of 3.1 Å for the tri-snRNP core, 4.3 Å for BRR2, 3.3 Å for the U4/U6 snRNP and 12 Å for the U2 snRNP. g, Map versus model FSC curves generated for the tri-snRNP core, BRR2 and U4/U6 regions of B-like using PHENIX mtriage. h-i, Structure of the CE B-like dimers (panel h) and CI B dimers²³ (panel i). Upper panels, overview of the 3D organization of the dimers. Middle and lower panels, expanded views of interface 1 and 2, respectively. Note that the view of the interfaces is from the top of the complexes shown in the upper panels. As in the CI hB dimer²³, the B-like dimer is organized in a parallel manner with U5 located at the bottom of both protomers. Both dimers contain an upper, poorly defined globular density. Based on the position of the thin density bridges adjacent to SF3B1^HEAT, in both dimers the upper globular density appears to contain the MINX exon and associated proteins, and may additionally contain loosely-associated U1 snRNP. The two protomers in the B-like and B dimers are connected in the middle by an interface involving SMU1 and its binding partner RED, the PRP6 N-terminal HAT domains, and SNU66 α−helix^200-219. The CI B dimers contain a large globular density element that is located at the very bottom, close to the exit site of the 5’ exon in each protomer, suggesting that this density element is comprised of the 5’ exons and associated proteins²³. Consistent with this idea, CE B-like dimers, which lack an upstream 5’ exon, do not possess this large globular density. This supports the idea that the poorly-defined globular densities indeed are comprised of exons and exon-interacting proteins. In B-like, the lower interface 2 is comprised of unassigned density that separates SNU114 and MFAP1 from one protomer with SNU114 and MFAP1 from the other protomer. The functional relevance of these interfaces, as well as dimer formation in general, is currently not known. j, Fit of the nucleotides and protein side chains shown in Fig. 2b (left) and 2d (right) into the B-like EM density. k, Schematic of the RNA-RNA interaction network in the B-like complex. Two copies of the 5’ss-containing RNA oligonucleotide (5’ss oligo), which is added in excess, base pair not only with U1 (disrupting the U1/5’ss helix), but also with the U6 snRNA at/near the ACAGA box and with U5 snRNA loop 1. The resulting extended U6/5’ss helix is not as long as the bona fide extended U6/5’ss helix that is formed in the human B complex^19,22. A dot between two nucleotides indicates that they do not base pair, but that a helix involving these nucleotides is formed (e.g., the extended U2/BS or U6/5’ss helices).