Extended Data Fig. 5: Cryo-EM and image-processing of the pre-B^5’ss and pre-B^{5’ss+ATPγS} complexes.

a, RNA composition of pre-B^5’ss complexes. Purified pre-B complexes were incubated with an excess of the 5’ss RNA oligonucleotide and subjected to glycerol gradient centrifugation. 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 three independent pre-B^5’ss purifications with similar results. b, Representative cryo-EM 2D class averages of the pre-B^5’ss dimers. c, Cryo-EM computation sorting scheme for pre-B^5’ss complexes. All major image-processing steps are depicted. Two major classes of the pre-B^5’ss dimers are detected, which are organized in an antiparallel manner and are highly similar to the class 1 and class 2 dimers of the CE pre-B complex (Fig. 1a and Extended Data Fig. 1). In the class 2 dimer, the structure of only one pre-B complex is well-defined, whereas in class 1 both protomers are well-defined. d, Local resolution estimation of the tri-snRNP core region of the pre-B^5’ss complex. e, Orientation distribution plot for the particles contributing to the reconstruction of the tri-snRNP core region. f, Fourier shell correlation (FSC) values indicate a resolution of 4.2 Å for the tri-snRNP core in pre-B^5’ss. g, Map versus model FSC curves generated for the tri-snRNP core region of pre-B^5’ss. h, Comparison of the structure of pre-B^5’ss complexes formed by incubating purified pre-B with the 5’ss oligo at 0 °C or 30 °C. An overlay of the EM densities (low-pass filtered to ~20 Å resolution) is shown on the right. Pre-B^5’ss complexes with a nearly identical 3D structure were obtained if incubation with the 5’ss oligo was carried out at 0 °C or 30 °C. i, RNA composition of pre-B^{5’ss+ATPγS} complexes. Purified pre-B complexes were incubated with an excess of the 5’ss RNA oligonucleotide followed by ATPγS, and then subjected to glycerol gradient centrifugation. 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 two independent pre-B^{5’ss+ATPγS} purifications with similar results. j, Representative cryo-EM 2D class averages of the pre-B^{5’ss+ATPγS} dimers. k, Cryo-EM computation sorting scheme. All major image-processing steps are depicted. l, Local resolution estimation of the tri-snRNP core region of the pre-B^{5’ss+ATPγS} complex. m, Orientation distribution plot for the particles contributing to the reconstruction of the tri-snRNP core region. n, Fourier shell correlation (FSC) values indicate a resolution of 3.1 Å for the tri-snRNP core and 4.0 Å for BRR2. o, Map versus model FSC curves generated for the tri-snRNP core and BRR2 regions of pre-B^{5’ss+ATPγS}. p, Fit of two monomeric pre-B^{5’ss+ATPγS} complex molecular models into the EM density of the pre-B^{5’ss+ATPγS} dimer. As in B-like, the two pre-B^{5’ss+ATPγS} complexes are organized in a parallel manner, with their U5 Sm cores at the bottom. In addition, there is an upper globular density that, like in the B-like dimers, likely contains the exons and associated proteins. However, consistent with the absence of the B-specific proteins, the middle interface present in the B-like dimers that is formed in part by SMU1 and RED, is missing. Furthermore, the lower bridge that contained MFAP1 in B-like complexes, now appears to form mainly between the PRP28 helicase of each protomer. q, Comparison of the structure of complexes formed by incubating purified pre-B with the 5’ss oligo plus ATP (pre-B^5’ss+ATP) or ATPγS (pre-B^{5’ss+ATPγS}). An overlay of their EM densities is shown on the right, confirming that they possess a highly similar structure.