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Figure 6
Interaction between ScpAN and the Smc neck derived from T. onnurineus. (a) Pull-down assays using the indicated proteins and Ni–NTA resin. (His)10-GST-ToScpAN and (His)10-GST-BsScpAN were incubated with ToSmcHd-CC80 and BsSmcHd-CC30, respectively, in a 1:1 molar ratio (20 µM) and were applied to Ni–NTA resin. Proteins retained on the resin after washing with 30 mM imidazole solution were visualized on an SDS–polyacrylamide gel. (b) Modeling the interaction between ToScpAN and the ToSmc neck. ToScpAN was structurally aligned with BsScpAN bound to BsSmcHd-CC30 (PDB entry 3zgx; Bürmann et al., 2013BB5). Sequence alignments were performed using Clustal Omega (Sievers et al., 2011BB30). The black arrows indicate the residues involved in interaction at the binding interface between BsScpAN and BsSmcHd-CC30. Manual adjustment according to the structural alignment was unnecessary. (c) Cysteine positions introduced for BMOE cross-linking experiment. Based on the model of ToScpA–ToSmcHd-CC80 (left), Glu69 on ToScpA and Gln185, Gln994 and Ala1110 on ToSmcHd-CC80 were selected as the mutation sites (right three panels; cyan sticks). (d) BMOE cross-linking. The cross-linked bands are labeled from 1 to 3, and the identity of these bands were deduced based on the GFP signal (right) and mass spectrometry (Supplementary Table S3).

Volume 7| Part 2| March 2020| Pages 193-206
ISSN: 2052-2525