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1.
Figure 3

Figure 3. Low-Level Glutaraldehyde Crosslinking Further Stabilizes Hsp90:Hop. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

In (A) is a micrograph of S-S Hsp90:Hop with boxed particles in a compact (white) and extended (orange) arrangement. In (B) is a micrograph of S-S Hsp90:Hop following an incubation 0.005% glutaraldehyde, showing homogeneous compact particles (box size = 300 Å). Hsp90:Hop conformational equilibrium model is shown (C) where alternate Hsp90 and Hop interactions result in an extended state or a compact, V-shaped state that is observed in the reconstruction and stabilized by glutaraldehyde crosslinking.

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
2.
Figure 5

Figure 5. Hop Makes Extensive, Independent Interactions with Hsp90. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

Density corresponding to two Hop molecules (pink) is shown in (A) revealing extensive interactions with Hsp90 and significant density projecting out across the interdimer cleft. TPR1 and 2A domains fit manually into EM density (C), show TPR1 is potentially accessible to Hsp70, extending between the Hsp90 monomers (grey) and TPR2A interacting along the Hsp90 MD-CTD junction. EM density is contoured to a 300 kDa mw.

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
3.
Figure 2

Figure 2. Negative-stain Reconstruction of the Disulfide Crosslinked Hsp90:Hop Complex. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

In (A) are reference-free (RF) class averages (box size = 300 Å) and initial model. In (B) are example single particles (SP), corresponding class averages and 3D images with projection direction (φ, θangles) shown. The final Hsp90:Hop negative-stain 3D model filtered to 25 Å is shown in (C) with approximate dimensions. For comparison, similarly oriented views of the apo E. coil Hsp90 crystal structure (Shiau et al., 2006) are shown (D), colored by domains: NTD (blue, residues 1–227), MD (green, residues 233–489), and CTD (sand, residues 501- 624).

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
4.
Figure 7

Figure 7. Hsp90:Hop Complex is Optimally Poised for Client Delivery by Hsp70. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

In (A) hydrophobic surface regions (raspberry) are mapped onto the extended Hsp90 solution conformation (Krukenberg et al., 2008) and compared to the Hop-stabilized conformation revealing a convergence of hydrophobic patches in the interdimer cleft in the Hop-stabilized state. In the model for client delivery (B) Hop first binds, stabilizing the Hsp90 client-loading conformation observed in our cryo-EM structure. The Hsp70:ADP:client complex then binds, interacting with TPR1 of Hop and Hsp90 in an arrangement similar to our 2D averages of Hsp90:Hop:Hsp70 and releases the client to hydrophobic residues in the interdimer cleft. Once Hop and Hsp70 release and ATP binds the Hsp90 NTDs dimerize, forming the closed state (Ali et al., 2006). ATP hydrolysis and release of the activated client then occur and Hsp90 is proposed to cycle through a compact ADP state (Southworth and Agard, 2008).

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
5.
Figure 4

Figure 4. Alternate Hsp90 Conformation of the 15 Å Cryo-EM Reconstruction of Apo Hsp90:Hop. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

Reference-free cryo-EM averages aligned to projections of the final model are shown (A) along with corresponding class averages (box size = 250 Å). In (B) the resolution of the reconstruction is estimated to be 15 Å by the Fourier shell correlation method (FSC). In (C) is the final cryo-EM reconstruction, filtered to 15 Å. The apo E. coli Hsp90 crystal structure (Shiau et al., 2006) fit into the EM density (D) shows the NTDs (blue) projecting out of the density while the MD-CTD (orange) arrangement matches the reconstruction. An improved fit is shown (E) where the NTD-MD angle from the yeast Hsp90:p23:AMPPMP closed structure (Ali et al., 2006) is mapped onto the apo structure.

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
6.
Figure 6

Figure 6. Hsp70 Binds Hsp90:Hop, Forming an Asymmetric Client-Loading Complex. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

In (A), a single Hsp70 binds either wt (green) or S-S Hsp90:Hop (red) forming a stable complex. Wt Hsp90:Hop:Hsp70 is measured to be 310 kDa (300 kDa calculated for 2Hsp90:1Hop:1Hsp70) compared to the S-S Hsp90:Hop:Hsp70 complex measured to be 370 kDa (calculated as 370 kDa for 2Hsp90:2Hop:1Hsp70) and wt Hsp90:Hop alone (black), measured to be 230 kDa (233 kDa, calculated). In (B) are reference-free averages of the S-S Hsp90:Hop:Hsp70 complex. In (C) is SDS-PAGE analysis of uncrosslinked (lanes 1–4) and glutaraldehyde crosslinked (lanes 5–7) Hsp90 alone and wt and S-S Hsp90:Hop. For size comparison, non-reduced S-S Hsp90:Hop, (runs as a 148 kDa dimer in SDS), was run (lane 4). In (D) are reference-free averages of the S-S Hsp90:Hop tetramer complex aligned to reference-fee averages of the wt trimer complex. In (E) are top-down views of 3D negative-stain reconstructions of the S-S tetramer and wt trimer complex with two rounds of refinement and no imposed symmetry. The arrow indicates a lobe of Hop density that is absent in the trimer complex. Box sizes (B, D) are 300 Å.

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.
7.
Figure 1

Figure 1. Specific Disulfide Crosslinking Stabilizes 300 kDa Human Hsp90:Hop Tetramer Complex. From: Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex.

Domain map of Hop (A) with a TPR crystal structure (Scheufler et al., 2000) shown for TPR1 (green), TPR2A (raspberry) and TPR2B (lilac). (B) SEC-MALS analysis of wt Hsp90 alone (red) and incubated with Hop (blue). Mw’s determined to be 170, 65, and 230 kDa for Hsp90, Hop and Hsp90:Hop, respectively. In (C) a 300 kDa tetramer complex is determined when Hsp90 and Hop containing cysteine mutations (733C and T260C, respectively) are incubated together and analyzed by SEC-MALS. Mw’s determined from the Raleigh ratio, measured by static light scattering, and the protein concentration (right Y-axis), measured by the differential refractive index. Calculated mw’s in kDa are: 170 (Hsp90 dimer), 63 (Hop), 233 (2Hsp90:1Hop), and 296 (2Hsp90:2Hop). (D) Fractions collected from the Hsp90:Hop peak in (B) were analyzed by SDS-PAGE in the absence and presence of 5 mM DTT. In (E) the TPR 2B crystal structure is shown with the bound MEEVD peptide (black) and the approximate location of the 733C:T260C disulfide.

Daniel R. Southworth, et al. Mol Cell. ;42(6):771-781.

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