Extended Data Figure 2: Cryo-EM and image processing of the human C* complex.

a, Typical cryo-EM raw image of human C* spliceosomes recorded with a Titan Krios (FEI Company) electron microscope at a nominal magnification of 88,000× with a Falcon II direct electron detector resulting in a pixel size of 1.59 Å per pixel. b, Several representative class averages showing different views of the C* particle after 2D classification. c, Euler angle distribution of all particle images that contributed to the final 3D map. The coordinates describe the ϕ and θ angles. Size and colour of the plotted dots indicate the number of particles at any given Euler angle. Although several sets of particle orientations dominated, an almost complete angular coverage was obtained. d, Computational sorting scheme. Imaged micrographs were first evaluated and sorted according to the quality of their Thon rings in local power spectra. Roughly 2.5 million particle images were then selected from the remaining micrographs. In a second sorting step, particle images were again discarded based on the quality of Thon rings in classified, local power spectra. Following evaluations in Fourier space, particles were then excluded according to multiple rounds of 2D classifications. The remaining 1,708,164 particle images were split into seven subsets of approximately 244,000 particles, and each subset again separated into six classes by 3D classification in RELION. One of the results is shown as an example. Images contributing to the best defined spliceosome structure (around 23%) were then merged again into one particle subset. Further rounds of 3D classification in RELION led to a refinement of particle homogeneity within the final subset. The latter, now consisting of 136,534 particles was refined to a structure at 8.4 Å resolution without masking. The final structure at 5.9 Å resolution was obtained by applying a soft mask during the final steps of the refinement process. To evaluate details at the core of the C* EM density, the unfiltered map obtained after the final 3D refinement was low-pass filtered to 4.5 Å and sharpened using a B-factor of −350 in RELION. e, Local resolution plot reveals a resolution distribution from approximately 4.5 to 10 Å with some less well-defined parts at the periphery of the complex. Higher resolution regions (in blue, up to 4.5 Å resolution) were obtained for the centrally located catalytic core of the spliceosome. f, Fourier-shell correlation function of two independently refined half data sets indicates a global resolution of 5.9 Å for the masked C* spliceosome comprising around 80% of the visible density of the whole spliceosome with respect to the unmasked density volume.