Fig. 5: Overall structure modelling of the PIF4^bHLH–DNA complex.

a,b, Domain architecture of PIF4 (a) and alignment of bHLH domain among PIF1, 3, 4, 5, 6, 7 and 8 proteins (b) of Arabidopsis. The dotted box on PIF4 indicates the protein boundaries used for structure modelling and gel-shift assays. c, Size-exclusion chromatography analysis of PIF4^bHLH WT and mutant (Mu1) proteins. PIF4^bHLH domains were fused with an MBP tag at the N terminus. The x axis and y axis indicate the elution volume and protein absorption at 280 nm. The WT PIF4^bHLH and Mu1 proteins are coloured in green and pink, respectively. a.u., arbitrary units. d, Eluted protein samples from the same peak fractions of WT PIF4^bHLH and Mu3 proteins from c, were subjected to SDS–PAGE followed by Coomassie blue staining. Size-exclusion chromatography analysis in d was performed twice with similar results. e, Modelled structure of the DNA-bound PIF4^bHLH homodimer and zoom-in of the predicted PIF4^bHLH residues that interact with DNA are coloured in blue (Interface 1) and pink (Interface 2). f, Gel-shift analysis of the interactions between PIF4^bHLH WT, Mu1 and Mu2 proteins and DNA probes (as described in Fig. 4f). g, Modelled structure of the DNA-bound PIF4^bHLH homotetramer and a zoom-in of the predicted PIF4^bHLH residues that contribute to tetramer formation are coloured in black (Interface 3) and red (Interface 4). h, Gel-shift analysis of the interactions between PIF4^bHLH WT, Mu3 proteins and DNA probes (as in Fig. 4f). Alignment of the two predicted DNA-interacting interfaces and the two protein-interacting interfaces among the Arabidopsis PIF proteins and residue mutations of those interfaces on PIF4^bHLH are shown in b. EMSA assays in f and h were performed three times with similar results.

Source data