Extended Data Fig. 9: CLOCK-BMAL1 engages in protein–protein interactions on tandem E-boxes.
From: Cooperation between bHLH transcription factors and histones for DNA access
a, Representative cryo-EM micrographs from two collected datasets (10,693 micrographs, dataset 1; 14,572 micrographs, dataset 2) denoised with Janni. b,. Movies were motion-corrected in RELION v.3, then CTF correction, particle picking as well as multiple rounds of 2D classification were performed in cryoSPARC v.3.1. Particles from dataset 1 were used for 3D reconstruction and after refinement, were transferred into RELION. They were used as an input model for 3D classification of dataset 2 in RELION. After multiple rounds of 3D classification and refinement both datasets were merged and subsequent 3D classification with signal subtraction and 3D Flex reconstruction yielded a homogeneous subset of particles. The boxes defined by the dashed line indicate the good models and set of particles used for the following step in the data processing workflow. c, Gold-standard FSC curve for the 3.8 Å resolution map highlighted by the red box in b. d, Local-resolution filtered map (MonoRes) for the 3.8 Å resolution map highlighted by the red box shown in b. e, Angular distribution for the particles leading to the 3.8 Å resolution map. f, Gold-standard FSC curve for the 6.1 Å resolution map highlighted by the blue box shown in b. g, Angular distribution for the particles leading to the 6.1 Å resolution map. h, Local-resolution filtered map (MonoRes) for the 6.1 Å resolution map highlighted by the blue box shown in b. i, Internal CLOCK-BMAL1 in Por map overlays well with the single CLOCK-BMAL1 heterodimer bound in the NCPSHL+5.8-W601 structure. j, F-alpha PAS-A helix of BMAL1 interfaces with the histones when CLOCK-BMAL1 binds at SHL-6.2. k, Sterically incompatible cross-links when mapped to the PAS domains of a single CLOCK-BMAL1 heterodimer. l, Map fit of tentative tandem CLOCK-BMAL1 model best compatible with cross-linking and cryo-EM data. The map is at 0.005. m, Tentative CLOCK-BMAL1 tandem model with putative inter-CLOCK-BMAL1 and CLOCK-BMAL1-histone cross-links mapped. Putative inter-CLOCK-BMAL1 cross-links would be sterically incompatible when mapped to a single heterodimer (see k). n, Distance distribution of cross-links mapped to the tandem CLOCK-BMAL1 model shown in panel m. o, Molecular mass distribution histogram of CLOCK-BMAL1-NCPSHL+5.8 (single E-box) and CLOCK-BMAL1-NCPSHL+5.8-tandem (2 E-boxes with 7-bp spacing as in the Por structure but with a 601 sequence). The tandem E-box arrangement increased the amount of CLOCK-BMAL1-bound complex from 19% to 51%. p, Molecular mass distribution histogram of CLOCK-BMAL1-NCPPor. q, Western blot comparing BMAL1 protein expression across reconstituted cell lines. The blot is representative of 3 biological replicates. r, GST pull-down assay performed by incubating His–GST-tagged CRY-binding domain of Per2 (His–GST-PER2-CBD) as bait with the prey proteins: photolyase homology region (PHR) of CRY1 and CLOCK-BMAL1 wild-type or mutant constructs. CLOCK and BMAL1 bHLH PAS-AB both are of very similar molecular weight, therefore, appear as one single band. The gel shown is representative of n = 3 independent experiments.
