Fig. 3: Reconstitution of the WAVE Shell Complex (WSC).

a In vitro translation of plasmids encoding subunits encoding the WRC with NHSL1 full length (FL) or indicated fragments in an untagged form or tagged with HA or GFP, as indicated. Immunoprecipitation targeting the HA tag pulls down the WSC since the HA tagged NHSL1 N-terminal fragment (1–123), containing the WHD, excludes WAVE2. In contrast, the NHSL1 C-terminal fragment (124–1639) associates with the WRC containing WAVE2. b Competition experiments using in vitro translation. WAVE2 displaces NHSL1 WHD for binding the shell subunits CYFIP1, NCKAP1, ABI1, and BRK1, but NHSL1 WHD does not displace WAVE2. c Reconstitution of recombinant WSC and WRC. Coomassie-blue stained SDS–PAGE gel, where each lane contains 3 pmol of purified complex. d Gel filtration chromatography of recombinant WSC and WRC using a 24 ml Superdex 200 column. Vo void volume. Recombinant complexes are pure, assembled and do not aggregate. e Electron microscopy of recombinant WSC and WRC after negative staining. Representative raw micrographs, 2D class averages and number of particles used to derive the class averages are shown. Note the very similar shapes of the two complexes, particularly on the first three class averages that were selected to display similar orientations. Experiments were performed once in a and e for the WSC, twice in e for the WRC and three times in c and d with similar results.