Fig. 4

Crystal structure of TRIM25 CC-PRYSPRY. a The TRIM25 CC dimer (in dark and light blue) with two PRYSPRY domains bound at either end in green cyan. There is no electron density for the linker connecting the CC and PRYSPRY domains. The distance between the N-termini of the PRYSPRY domains and C-terminus of one CC chain is indicated. b Close-up view of the interface between the CC and PRYSPRY domain. Residues described in the text are shown as ball and stick. c Ubiquitination assays of the tandem CARDs of RIG-I with TRIM25 to test the importance of Y463 and Y476 in stabilising the observed CC-PRYSPRY arrangement and promoting substrate ubiquitination. FLAG-RIG-I-2CARD and TRIM25 were co-transfected into HEK293T cells and WCLs were subjected to IP with anti-FLAG beads and immunoblotted with α-ubiquitin and α-FLAG antibodies. The asterisk indicates an unspecific band. d Comparison of the positions of the PRYSPRY domain (green cyan) and the NS1 ED (red), highlighting the changes in the position of the linker connecting the CC and PRYSPRY domain, induced by binding to NS1