(A) Fit of the MED23 X-ray structure into the corresponding portion of mMED Tail cryo-EM map. (B) Comparison between cryo-EM maps of the mMED Tail (large segment in solid gold, small segment in solid salmon, and MED23 in transparent light purple) and the Sc Tail (transparent blue). (C) Subunit organization and interaction of the mMED Tail. The large Tail segment includes MED16 (gold), MED23 (light purple), MED24 (N-terminal portion in tan; C-terminal portion in dark green) and MED25 (red). The smaller Tail segment (in salmon) includes MED15, and MED27–30. The large Tail segment interacts (through the C-terminal portion of MED24) with MED1 in the Middle module (blue mesh) and with the C-terminus of MED14 (light green mesh). The smaller mMED Tail segment also interacts with the MED14 C-terminus and with the lower part of the Head (red mesh), including the MED18-MED20 Head jaws. The general position of MED26 from EM analysis of human Mediator is indicated. (D) Module assignment for mMED subunits based on EM analysis results. MED14, which functions as a central scaffolding subunit, was not assigned to a specific module.