Extended Data Fig. 1: Purification and cryo-EM imaging of the human U5 snRNP complexes.
From: Structural basis of human U5 snRNP late biogenesis and recycling
a. Endogenous CD2BP2 is detected by western blot in nuclear and cytoplasmic extracts (NE and CE) from K562 wild-type or K562 GFP-CD2BP2 overexpressing cells (representative image, n = 4). Note that the western blot band for CD2BP2 migrates at a higher molecular weight in CE compared to NE fractions, indicating that post-translational modifications could regulate CD2BP2 activity. Consistent with this, CD2BP2 has been reported to be phosphorylated, which may influence its interactions with the U5 snRNP41. b. GFP-CD2BP2 purifications from nuclear or cytoplasmic extracts (NE and CE) yield U5 snRNPs of similar protein composition (n = 2). c. Purification scheme (top) and SDS-PAGE analysis (bottom) of the human U5 snRNP complexes from K562 cells. The most abundant proteins in the samples are annotated according to mass spectrometry analysis of SDS-PAGE gel slices. The asterisk annotates contaminants (n = 9). d. Denoised cryo-EM micrographs of U5 snRNP complexes. The dataset contained 21,572 micrographs. Scale bar, 300 Å (n = 1). e. Cryo-EM 2D class averages of U5 snRNP complexes in State I, II, III, IV. The particles subsets for the shown States I-IV 2D class averages were obtained from U5 snRNP image processing shown in Extended Data Fig. 2. Scale bar, 100 Å.
