Sirt6/ADP-ribose crystals were grown from 1.6 M (NH4)2SO4, 10% PEG 400, and Bis-Tris buffer pH 5.7 with 10 mg/ml Sirt6(13–308) and 10 mM ADP-ribose by the hanging drop vapor diffusion method at 20 °C17 . The Sirt6/ADPr/quercetin and Sirt6/ADP-ribose/cyanidin complexes were obtained by soaking Sirt6/ADP-ribose crystals with 40 mM compound for one week, and the Sirt6/ADP-ribose/CG complex by soaking with 1 mM CG overnight. The structure in complex with isoquercetin was produced by transferring crystals in a new drop containing 1.6 M (NH4)2SO4, 10% ethylene glycol, and Bis-Tris buffer pH 5.7 with 40 mM isoquercetin and incubation for one week. A solution of reservoir supplemented with 20% ethylene glycol, 10 mM compound and 2 mM ADP-ribose was used as cryoprotectant.
Sirt2/ADP-ribose crystals were grown in hanging drops at 20 °C with 14% PEG 10.000 and 0.1 M ammonium acetate pH 5.8 as reservoir solution40 (link). The protein solution contained 13 mg/ml Sirt2 (55–356) and 20 mM ADP-ribose. The Sirt2/ADP-ribose crystals were soaked with 40 mM quercetin for 3 days. Crystals were then transferred to a drop of reservoir supplemented with 20% glycol, 10 mM quercetin and 2 mM ADP-ribose before flash freezing in liquid nitrogen.
Diffraction data were collected at 100 K at BL14.1 operated by Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany)41 (link). Diffraction data were processed with the X-ray Detector Software (XDS) using XDSapp42 (link),43 (link). The Sirt6 crystals were detected to be twinned through L-tests in POINTLESS44 (link). Structures were solved by molecular replacement phasing with Phaser45 (link) from the CCP4 software suite46 (link), using the twinned data and a Sirt6/ADP-ribose structure (PDB code 3K35)31 (link) as a search model for the Sirt6 complexes and Sirt2/1,2,4-Oxadiazole/ADP-ribose structure (PDB code 5MAR)47 (link) for the Sirt2 complex. The structures were manually rebuilt in COOT48 (link) and refined with Refmac49 (link). The refinements were done with the amplitude-based twin refinement option and yielded twin fractions of 22–44% (Table1 ). Structure figures were generated with PyMOL (Schrödinger, LLC; https://pymol.org/2/ ).
Sirt2/ADP-ribose crystals were grown in hanging drops at 20 °C with 14% PEG 10.000 and 0.1 M ammonium acetate pH 5.8 as reservoir solution40 (link). The protein solution contained 13 mg/ml Sirt2 (55–356) and 20 mM ADP-ribose. The Sirt2/ADP-ribose crystals were soaked with 40 mM quercetin for 3 days. Crystals were then transferred to a drop of reservoir supplemented with 20% glycol, 10 mM quercetin and 2 mM ADP-ribose before flash freezing in liquid nitrogen.
Diffraction data were collected at 100 K at BL14.1 operated by Helmholtz-Zentrum Berlin (HZB) at the BESSY II electron storage ring (Berlin-Adlershof, Germany)41 (link). Diffraction data were processed with the X-ray Detector Software (XDS) using XDSapp42 (link),43 (link). The Sirt6 crystals were detected to be twinned through L-tests in POINTLESS44 (link). Structures were solved by molecular replacement phasing with Phaser45 (link) from the CCP4 software suite46 (link), using the twinned data and a Sirt6/ADP-ribose structure (PDB code 3K35)31 (link) as a search model for the Sirt6 complexes and Sirt2/1,2,4-Oxadiazole/ADP-ribose structure (PDB code 5MAR)47 (link) for the Sirt2 complex. The structures were manually rebuilt in COOT48 (link) and refined with Refmac49 (link). The refinements were done with the amplitude-based twin refinement option and yielded twin fractions of 22–44% (Table
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