Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Mapping the interactions in human R2TP core components. a A cartoon for sequence and domains of the components of the human R2TP complex. b GST pull-down experiments depicting the interactions between the several regions in RPAP3 and PIH1D1. FL stands for full length, CS for the CS domain in PIH1D1, and MW for molecular weight markers. Be aware that for simplification, several PIH1D1 and RPAP3 constructs are indicated within the same lines on top of the gel. Some minor contaminants are present in some of the samples. c Pull-down experiments showing that removal of residues 401–420 from an RPAP3 construct eliminates the interaction with the CS domain in PIH1D1. d Pull-down experiments demonstrating the interaction of RPAP3–RBD with RUVBL2. This interaction is not affected when the DII domains in RUVBL2 are removed

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Sequencing, Molecular Weight, Construct

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of RUVBL1–RUVBL2 and the RBD domain. a 2D averages corresponding to top and side views obtained from cryo-EM images of the RUVBL1–RUVBL2 preparation in an ADP-containing buffer. b After incubation with RPAP3 430–665 , RBDs decorate the ATPase side of both RUVBL rings without disrupting the dodecamer, and a representative 2D average of the complex between RUVBL1–RUVBL2 and RBD is shown. At the right end of the panels, one view of the 3D structure of RUVBL1–RUVBL2–RBD complex with RBD domains in yellow. Note that one of the RBD domains in the bottom ring is less visible at the threshold used for rendering, probably reflecting variable occupancy. Also, the scale of the 3D structure has been enlarged with respect to the 2D average, for clarity

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Cryo-EM Sample Prep, Imaging, Incubation

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: Cryo-EM imaging of the R2TP complex. a Pull-down experiments showing the in vitro reconstitution of R2TP. M indicates molecular weight markers. b Purification of RUVBL1–RUVBL2 and PIH1D1–RPAP3 sub-complexes, used for the reconstitution of R2TP for cryo-EM. M indicates molecular weight markers. c Two representative side view averages of R2TP. RUVBL1–RUVBL2 rings are decorated by the RBD at the top (labeled with white arrows). A blurred and very flexible region locates at the bottom of the ring. d A representative side view average of R2TP reconstructed using the RPAP3–ΔNT–PIH1D1 sub-complex and RUVBL1–RUVBL2. Flexible regions at the bottom end of R2TP disappear when the N-terminal half of RPAP3 is removed, but dodecameric RUVBL1–RUVBL2 is disrupted. e 3D structure of R2TP obtained applying 3-fold symmetry. RBDs are bound to RUVBL1–RUVBL2 but the flexible regions in the complex are not resolved. Scale bar, 2.5 nm

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Cryo-EM Sample Prep, Imaging, In Vitro, Molecular Weight, Purification, Labeling

Journal: Nature Communications

Article Title: RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex

doi: 10.1038/s41467-018-03942-1

Figure Lengend Snippet: A cartoon for the structural and functional model for R2TP. a Human R2TP. HSP90 dimers can engage with each R2TP complex with sufficient conformational flexibility to reach and act in a diversity of client proteins. Up to 3 RBDs serve to anchor 3 RPAP3 to the RUVBL1–RUVBL2 scaffold, whereas a central segment of RPAP3 helps to recruit PIH1D1. The number of RPAP3 molecules per RUVBL ring in vivo is not known, and two options are shown in the figure. A long and poorly structured link between the RBD and TPR domains in RPAP3 results in substantially conformational flexibility of the TPR regions. For simplicity, although 3 RBDs are bound to the RUVBL ring, only 2 RPAP3s are shown bound to HSP90 in the cartoon. b Yeast R2TP. Conformational adaptability of yeast R2TP is limited to the flexibility of the C-terminal tails in Hsp90. Only one Hsp90 binds each R2TP.

Article Snippet: The RPAP3 full length (FL) was purchased from GenScript.

Techniques: Functional Assay, In Vivo

Journal: Biochimica et biophysica acta

Article Title: Inhibition and conformational change of SERCA3b induced by Bcl-2

doi: 10.1016/j.bbapap.2016.09.004

Figure Lengend Snippet: ATP2A3 and GST were used as described in 2.12.2. First, we quantified recombinant GST and used it as a standard to determine the concentration of ATP2A3. In the final step, we used ATP2A3 as a standard to quantify SERCA3b in microsomal fractions. Top: 2 µg, 4 µg, 5 µg and 6 µg of microsomal proteins were loaded onto the gel, along with different amounts of ATP2A3, and Western blot analysis was done with the anti-ATP2A3 antibody. Bottom: A calibration curve was generated by densitometry using relative peak area of ATP2A3 as explained in 2.12.2 (For ATP2A3 amounts >0.45 µg, we observed signal saturation in the Western blot; these data points were excluded from the calibration).

Article Snippet: Partial recombinant protein of SERCA3 with a GST tag at N-terminal, ATP2A3, (catalog no. H00000489-Q01) was purchased from (Abnova, Taiwan).

Techniques: Recombinant, Concentration Assay, Western Blot, Generated