Extended Data Fig. 1: Cryo-EM analysis of C. elegans spliceosomes. | Nature

Extended Data Fig. 1: Cryo-EM analysis of C. elegans spliceosomes.

From: Mechanism for the initiation of spliceosome disassembly

Extended Data Fig. 1

a. Schematic of purification of spliceosomes from C. elegans. The endogenous locus of PRP19 was tagged with am N-terminal FLAG-tag using CRISPR/Cas9 and extract was prepared from ~12 million adult worms. After immunopurification (IP) and elution with FLAG peptide, spliceosomes were further purified via a sucrose gradient. b. Coomassie-stained SDS-Poly-Acrylamide Gel (SDS-PAGE) of gradient-purified Ce spliceosomes. This experiment was performed seven times. c. Denoised cryo-EM micrograph of gradient-purified and crosslinked Ce spliceosomes imaged on a Titan Krios with a K3 detector. d. 2D class averages from the dataset. e. Abundance of ILS subunits in gradient-purified sample measured by mass spectrometry. For this analysis we quantified absolute protein abundances by integrating the protein peptide peaks and normalizing to the protein length using iBAQ69, which were then normalized to PRP8. The labels next to the bars indicate how many peptides were identified for each subunit and which percent of the sequence was covered. f. Schematic of the data analysis pipeline. Stringent classification of ~4 million single particle images revealed the ILS′ (~85–90% of ILS particles) and ILS″ (~10–15% of ILS particles, see Supplementary Fig. 1) as the major PRP19-containing spliceosomes populations in Ce extract. Extensive focused refinements of each state yielded a total of 27 maps, revealing the ILS′ and ILS″ in unprecedented detail and facilitating the building of high-quality structural models. For details, see Extended Data Fig. 2 and Supplementary Figs. 1, 2. g. Sequence conservation plot of ILS subunits between human and C. elegans (Ce), and human and Saccharomyces cerevisiae (Sc) shows a highly conserved ILS protein composition between human and Ce.

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