Extended Data Fig. 1: Sample Preparation.
From: Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor
a, The in vitro reconstituted GR chaperone cycle. On the left, GR is active and able to bind ligand. Hsp70, aided by the co-chaperone Hsp40, engages GR and inhibits ligand binding. Hsp70 loads apo GR onto Hsp90 and Hop, which forms the ‘GR-loading complex’ (PDB ID 7KW7). Hsp70 and Hop are released, Hsp90 hydrolyzes ATP to fully close, and the co-chaperone p23 binds, forming the ‘GR-maturation complex’ (PDB ID 7KRJ). GR binds ligand in the transition from the GR-loading complex to the GR-maturation complex. In the maturation complex, GR is in a fully folded, native conformation and bound to ligand. Upon Hsp90 re-opening, GR is released from the complex to return to the cycle. b, Domain organization of the proteins in the GR:Hsp90:FKBP complexes and p23. c, Structural motifs of Hsp90, GR, and FKBP51/52. d, Coomassie-stained SDS-PAGE (4-12% acrylamide gel) with MBP-GR pulldown elutions from the in vitro reconstituted GR chaperone cycle. Lane 1- elution from MBP-GR pulldown for FKBP51-containing reaction; Lane 2- sample from Lane 1 after size exclusion chromatography (SEC) (e) and chemical crosslinking with 0.02% glutaraldehyde; Lane 3- elution from MBP-GR pulldown for FKBP52-containing reaction; Lane 4- sample from Lane 3 after SEC (e) and chemical crosslinking with 0.02% glutaraldehyde. This experiment was repeated 7 independent times with similar results. e, Size exclusion chromatography (SEC) profile of the elution from the MBP-GR pulldown. The green trace represents the SEC profile from the reconstituted GR chaperone cycle with FKBP51, while the blue trace represents the SEC profile from the reconstituted GR chaperone cycle with FKBP52. mAU = milli-absorbance units. Coomassie-stained SDS-PAGE (4-12% acrylamide gel) of the fractions from SEC corresponding to the GR:Hsp90:FKBP52 sample (top) or the GR:Hsp90:FKBP51 sample (bottom). Colors indicate which gel lanes correspond to specific regions of the SEC profile. Sample fractions from the region highlighted in purple were collected and used for cryo-EM data collection. This experiment was repeated 11 independent times with similar results. f, Representative electron micrograph for the cryo-EM dataset of the GR:Hsp90:FKBP52 complex (left) (−1.48 μm defocus) and GR:Hsp90:FKBP51 complex (right) (−2.23 μm defocus). A total of 11,162 and 26,413 micrographs were obtained, respectively.
