Extended Data Fig. 1: Cryo-EM sample preparation, imaging and processing for type I-F integration complex.
a, Sequence-level schematic of DNA used to assemble the integration complex. The length of each motif is listed. The latter two thirds of the CRISPR repeat and second spacer (grey dashed box) could not be resolved in the cryo-EM reconstruction. See also Supplementary Table 1. b, Size-exclusion chromatography (SEC) profile (Superdex 75 16/600, Cytiva) of IHF heterodimer purified as described in methods section, and SDS-PAGE gel (inset). c, SEC profile (Superdex 200 10/300, Cytiva) of Cas1–2/3 heterohexamer purified as described in methods section, and SDS-PAGE gel (inset). d, I-F integration complex was assembled from purified DNAs, IHF and Cas1–2/3 as described in the methods section, and the assembled complex was further purified by size-exclusion chromatography (SEC) (Superdex 200 10/300, Cytiva). Individual fractions were collected along the elution profile, and were concentrated and stored separately for further analysis and imaging. e, Individual SEC fractions were analyzed by SDS-PAGE to determine which fractions contained all the proteins necessary for a complete complex. f, Individual SEC fractions were phenol-chloroform extracted, and the aqueous layer was analyzed by Urea-PAGE to determine which fractions contained all four DNA strands necessary for a complete complex. The fraction chosen for cryo-EM analysis is indicated with a dotted purple box. g, Image processing pipeline for a small subset of 10,740 total micrographs for the type I-F integration complex, to generate an initial model for template picking. Scale bar represents 100 nm. h, Final image processing pipeline for the type I-F integration complex. i, Viewing direction distribution plot depicting particle orientations present in final reconstruction. More populated views are shown in red, and less populated views are shown in blue. j, 3D Fourier Shell Correlation (3DFSC) of the final I-F integration complex reconstruction. The global resolution at 0.143 is indicated by a dashed line, 3.48 Å. k, Local resolution estimation of the cryo-EM reconstruction calculated by cryoSPARC81. The purification of proteins, assembly of the integration complex, and analysis of these samples by SDS-PAGE or Urea page was performed once. Micrographs were collected on two separate occasions with similar results.
