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. 2023 Jul 19;6(9):e202201790. doi: 10.26508/lsa.202201790

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© 2023 Karl et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure S4. — (A) In trans complementation setup: SAM-key construct with the SAM-key (red) is added to Fun30∆SAM, where domain architecture shows CUE domain (rosy brown) and SNF2-type ATPase domain consisting of N-terminal lobe (beige), protrusion I (orange), insertion II (beige), and C-terminal lobe (beige). Color scheme used throughout the figure. (B) AlpaFold2 multimer model of Fun30∆SAM and SAM-key shows the interaction surface between SAM-key and protrusion I as in the full-length protein. Model shows high confidence in predicted structured regions (colored). Box indicates region of zoom in the SAM-key (red) with predicted interaction to the protrusion I (orange). Amino acids contributing to the hydrophobic interaction surface are highlighted: I367, V370, I371, C374 of the SAM-key (red) and F754, F761, I764, F765, M793, and F797 of protrusion I (orange). (C) XL-MS of Fun30∆SAM and the SAM-key (25x molar excess) with BS-3 crosslinking verifying the AlphaFold2 multimer model. Left: crosslinks visualized on 2D constructs (unfiltered). Arches depict crosslinks, blue color within Fun30∆SAM, purple crosslinks within the SAM-key, green intramolecular crosslinks. Red indicates close proximity of the crosslinks indicating the probability of being intramolecular. Right: crosslinks mapped on 3D model (CUE + unstructured regions excluded from model). Crosslinks shown as connectors. Crosslinks in low confidence, unstructured regions were removed, only crosslinks within predicted structured regions ± two additional aa residues were considered. Blue crosslinks (n = 51) match the model with a length restriction for BS-3 of 35 Å. Grey crosslinks (n = 2) violate the threshold and are >35 Å. Both violating crosslinks were supposedly intermolecular SAM-key crosslinks likely because of the high concentration of the SAM-key construct used in this experiment. Below: SAM-key showed three intermolecular crosslinks with protrusion I, all matching the 35 Å distance constraint and confirming the SAM-key–protrusion I interface. (D) Histograms showing the distribution of crosslink lengths for Fun30 WT (left) and Fun30∆SAM (right). Line indicates the threshold for BS-3 of 35 Å.