Extended Data Fig. 5: Conformational variability of ACC dimers and density of the covalently linked biotin cofactor.

a, Negative stain electron microscopy 2D class averages of dephosphorylated human ACC in the absence of citrate show the protein in a variety of conformations without considerable populations of closed dimers. b, Negative stain image of ACC–citrate filaments obtained from dephosphorylated ACC in a buffer with 10 mM citrate. Arrows indicate rarely observed, residual, non-polymerized ACC (further analysed in c). c, Negative stain electron microscopy 2D class averages of residual, non-polymerized ACC dimers observed in the presence of citrate on micrographs of polymerized ACC–citrate filaments (see b). ACC-like classes are marked in red. A variety of elongated conformations can be observed. d, Streptavidin (SA) shift assay to determine biotinylation level of ACC. ACC and streptavidin were mixed in different ratios and the shift upon SDS-resistant binding of streptavidin to biotin was observed via SDS–PAGE. At higher excess of streptavidin, no unbound ACC is observed, indicating complete biotinylation of ACC. Two degradation products of ACC (indicated by asterisks) are observed, one of which also shows a band shift (*-SA) and thus contains the biotinylated site. Owing to the tetrameric nature of streptavidin, higher-order complexes are formed, which can also be observed on the gel. An uncropped image of the gel is shown in Supplementary Fig. 1. e, f, Density of the covalently linked biotin cofactor in the two active sites of the CT domain dimer. The main chain Cα position of the biotinylated lysine (residue 786) is indicated. Due to limited resolution, the cofactor was not modelled; its orientation is shown schematically. For clarity, parts of BCCP are not shown. The map is shown at contour level of 0.0238.