Extended Data Fig. 6: Cryo-EM analysis of human ILS spliceosomes.

a. Schematic of purification of TFIP11-bound spliceosomes from human cells. GFP-TFIP11 was overexpressed in K562 suspension cells and TFIP11-bound spliceosomes were purified from 30 L of suspension cell culture. After immunoprecipitation (IP) and elution with 3C protease, spliceosomes were further purified via a sucrose gradient. b. Coomassie-stained SDS-Poly-Acrylamide Gel (SDS-PAGE) of the TFIP11-GFP IP. Bands in the gel are labelled according to the molecular weight of the ILS subunits. This experiment was performed four times. c. Denoised micrograph of gradient-purified and crosslinked Hs ILS, imaged on a Titan Krios G4 with a Falcon 4i detector. d. 2D class averages from the dataset. e. Composite cryo-EM density, obtained from 14 local refinements and filtered by local resolution. Transparent density in the background shows a local refinement map (focused on PAXBP1) low-pass filtered with a gaussian filter with a sigma of three standard deviations. f. Model of the human ILS″, with disassembly factors shown as ribbons and spliceosome core proteins shown in addition as a transparent surface. A difference density, calculated by subtracting simulated model density (low-pass filtered to 20 Å) resolution) from experimental density (ILS consensus refinement map low-pass filtered with a gaussian filter with a sigma of three standard deviations) reveals additional density at the Ce ILS″ C19L1 CWFJ position.