Computational protocol: A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme

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Protocol publication

[…] USP12 and UAF1 crystals were obtained by sitting drop vapour diffusion experiment at 20 °C using equal ratio of protein and mother liquor solution. UAF1580 was setup for crystallization at 8 mg/ml and crystals were obtained in 20% PEG3350, 200 mM Tri-Sodium Citrate, Bis-Tris Propane pH 6.5. USP12FL-Ub-PRG/UAF1580 was setup at 5 mg/ml and crystals were obtained in 3.2% PEG4000, 0.1 mM MMT pH 6.5 and 0.1 mM TCEP. The crystals were cryo-protected by brief washing in mother liquor solution with 30% glycerol prior to flash freezing in liquid nitrogen. Diffraction data were collected at the Swiss Light Source beamline PXIII at 100 K.USP46-Ub-VME at 8 mg/ml was set up for crystallization by sitting drop vapour diffusion experiment at 20 °C. Crystals were obtained in 0.96 M Sodium Citrate pH 7.5 and 0.1 mM ZnCl2 with protein: precipitant ratio 1.5: 1. The crystals were cryo-protected by brief washing in mother liquor solution with 20% glycerol prior to flash freezing in liquid nitrogen. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) beamline ID14-1 at 100 K.Crystallographic data were processed with XDS () or iMOSFLM () and scaled using XSCALE () or Aimless from the CCP4 suite (). The USP46-Ub-VME structure was solved by molecular replacement using the USP2-Ub model (PDB 2IBI) in Phaser () followed by automated model building using ARP/WARP (). The UAF1 (9–580) structure was solved by molecular replacement (PDB-1VYH) in Phaser () followed by automated model building using ARP/Warp (). The USP12FL-Ub/UAF1580 structure was solved by molecular replacement using the USP46, UAF1580 and ubiquitin structures as search models in Phaser. All structures were refined by Phenix (), autoBUSTER (), Refmac (), PDB_REDO () and models were built using COOT (). Interface analysis was performed with PISA (). All structure figures were generated using PyMOL (Schrödinger, LLC). [...] USP12FL-Ub/UAF1FL purified from insect cells was concentrated up to 38 μM. The buffer used for the size exclusion chromatography was also used for SEC-SAXS experiment on the BM29 SAXS beamline at the ESRF. Following equilibration, 30 μl of the purified USP12FL-Ub/UAF1FL was loaded on the Superdex 200 5/150 GL (GE, USA) and 1500 successive 1 s frames were collected through the protein elution peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank (derived from the buffer run) was subtracted. Data were analysed using the ATSAS software package (). A moving set of 10 or 20 frames was analysed across the high-intensity peak to estimate molecular weight from various methods (DATPOROD (), excluded volume from DAMMIF model () and SAXS MoW2 ()) and fit the data to the crystallographic structures in CRYSOL (). Frames prior to 670 and frames latter than 910 were considered too noisy or weak for analysis. OLIGOMER () was used to perform a fit to the experimental curve from different possible models of the complex and determine volume fractions of each component. Several OLIGOMER runs were performed using the 2:1 complex, both 1:1 complexes and UAF1 alone as models in different combinations. […]

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