Structure and Function of CRSP/Med2 Dylan J Taatjes, Robert Tjian  Molecular Cell  Volume 14, Issue 5, Pages 675-683 (June 2004) DOI: 10.1016/j.molcel.2004.05.014

Figure 1 Purification and Subunit Composition of CRSP/Med2 (A) Purification scheme used for isolation of CRSP/Med2. (B) Silver stained, gradient (5%–15%) acrylamide gel of CRSP/Med2, purified as shown in (A). Polypeptides present in the complex are listed according to their molecular weight to the right of the silver stained bands. Where applicable, the nomenclature system proposed by Rachez and Freedman (2001) is used here and throughout the text. Additional protein bands corresponding to breakdown products of the GST-VP16 fusion protein are indicated with asterisks. (C) Western blot analysis of CRSP/Med2 against the subunits shown. Molecular Cell 2004 14, 675-683DOI: (10.1016/j.molcel.2004.05.014)

Figure 2 EM Analysis of CRSP/Med2 (A) Untilted electron micrograph of negatively-stained CRSP/Med2 sample at ×30,000 magnification shows uniform staining and a homogeneous field of particles. Scale bar, 800 Å. (B) Angular distribution of 2826 experimental single particle images comprising the final CRSP/Med2 data set. The size of each circle correlates with the number of experimental images representing that particular set of Euler angles (larger circle, more images). (C) Plot showing the Fourier shell correlation (FSC) function of two independent CRSP/Med2 reconstructions and the 3σ noise curve (noise). Resolution is estimated at 31 Å using the 0.5 correlation coefficient cutoff. (D) 3D reconstruction of CRSP/Med2 at 31 Å resolution. The structure is rendered to a mass of 1.0 MDa, the approximate molecular weight of the complex. Four different views of CRSP/Med2 are shown, resulting from rotations of the complex as indicated. Arrows (A)–(C) indicate prominent, solvent-exposed regions in the complex (see text). Scale bar, 75 Å. Molecular Cell 2004 14, 675-683DOI: (10.1016/j.molcel.2004.05.014)

Figure 3 Comparison of VP16-CRSP/Med2 and VP16-CRSP/Med Structures Side-by-side comparison of CRSP/Med2 and CRSP/Med reveals clear similarities in conformation. Structures are shown from two viewing angles, rotated as shown. Also evident is a defined region of missing protein density in the CRSP/Med2 complex, indicated by the arrows, which represents the location of Med220 (see text). The red “X” denotes an isolated region where protein density has apparently shifted in CRSP/Med2. Scale bar, 75 Å. Molecular Cell 2004 14, 675-683DOI: (10.1016/j.molcel.2004.05.014)

Figure 4 CRSP/Med2 Is a Promoter-Selective Coactivator In vitro transcription on chromatin–assembled templates. Purified and recombinant basal transcription factors (TFIIA, -IIB, -IID, -IIE, -IIF, -IIH, and RNA polymerase II) were added to assembled chromatin templates for each transcription reaction shown (see Experimental Procedures). Activators (VP16, SREBP-1a and Sp1, or VDR/RXR) and coactivators (CRSP/Med or CRSP/Med2) were added as indicated. (A and B) Both CRSP/Med and CRSP/Med2 activate transcription in response to VP16 (A) or SREBP/Sp1 (B) on chromatin templates. (C) CRSP/Med, but not CRSP/Med2, potentiates activator-dependent transcription in response to VDR/RXR. VDR ligand (1,25-dihydroxyvitamin D3) was added as shown. Molecular Cell 2004 14, 675-683DOI: (10.1016/j.molcel.2004.05.014)