Extended Data Figure 4 |. Characterization of DDB1-CDK12-cycK complex formation.

a, Schematic of the TR-FRET setup. Positions of the FRET donor (terbium-streptavidin (Tb)) and acceptor (_Alexa488SpyCatcher (A)) are indicated in the structural model. b, Titration of CDK12-_Alexa488cycK (0–3.75 μM) to 50 nM _terbiumDDB1 and 5 μM CR8 or DMSO (n=3). c, Counter-titration of unlabelled wild-type CDK12-cycK (0–10 μM) to 50 nM _terbiumDDB1, 500 nM CDK12-_Alexa488cycK and 12.5 μM CR8 (n=3). d, Counter-titration of unlabelled wild-type DDB1 (0–10 μM) to 50 nM _terbiumDDB1, 500 nM CDK12-_Alexa488cycK and 1 μM CR8 (n=3). e, Titration of CDK12(R965K)-_Alexa488cycK (wild-type sequence of canonical isoform of CDK12; 0–3.75 μM) to 50 nM _terbiumDDB1 and 5 μM CR8 or DMSO (n=3). The CDK12 K965R variant, which was used throughout our in vitro studies (see Methods), shows a binding affinity indistinguishable from that of wild-type CDK12 (residue distal from the interface with DDB1 and cycK). f, Isothermal titration calorimetry (ITC) experiment (n=2, additional replicates for this and following panels are provided in Supplementary ITC Data). Specifications of the titration are given in the panel. Asterisk marking the approximate K_apparent value denotes that the binding affinity was too high to allow confident fitting of the binding curve. g, ITC experiment as in f (n=2). h, ITC experiment as in f (n=1). i, ITC experiment as in f (n=3). j, ITC experiment as in f (n=3). k, ITC experiment as in f (n=1).