Cryo-EM Structure of RUVBL1-RUVBL2 Complex

For cryo-EM, we produced the human RUVBL1-RUVBL2 protein complex, as described previously (17 (link), 21 (link)). Affinity purification using a nickel column (His-tag purification) was followed by Sephacryl S300 (GE Healthcare) size-exclusion chromatography in a buffer containing 50 mM tris, 300 mM NaCl, and 1 mM dithiothreitol. The purified RUVBL1-RUVBL2 complexes used for cryo-EM contain a His-tag at the N terminus of RUVBL1, but the RUVBL2 subunit, with which PIH1D1 interacts, does not have a tag. For pull-down experiments, we cloned a RUVBL1-RUVBL2 complex without tag in RUVBL1 and a C-terminal strep-tag in RUVBL2 and purified it by affinity purification, followed by gel filtration.
We purified the RPAP3_400–665-PIH1D1 complex, as described previously (17 (link)). Briefly, we used glutathione S-transferase (GST)–tag affinity chromatography to bind GST-RPAP3_400–665, and we eluted the protein using 50 mM glutathione. We incubated the elution with PreScission protease (3C protease) at 4°C overnight to remove the tag, followed by a subsequent gel filtration chromatography using an S200 26/60 column. We reconstituted R2TP-ΔNT by mixing RUVBL1-RUVBL2 and RPAP3_400–665-PIH1D1 subcomplexes in a 1:4 molar ratio, followed by dialysis in 25 mM Hepes (pH 7.8), 130 mM NaCl, and 10 mM 2-mercaptoethanol–containing buffer containing 0.5 mM ADP (pH 7.0) at 4°C for 4 hours.