Computational protocol: An Ankyrin-repeat ubiquitin binding domain determines TRABID’s specificity for atypical ubiquitin chains

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Protocol publication

[…] TRABID AnkOTU was expressed as GST-fusion protein in E. coli and purified by affinity chromatography. After removal of the GST-tag by PreScission protease, anion exchange chromatography and gel filtration produced homogeneous protein that was crystallized at 3.5 mg ml-1. AnkOTU crystals grew from 150 mM NaCl, 100 mM NaOAc, 5 mM MgCl2, 50 mM MES [pH 5.9]. Crystals were soaked in mother liquor containing 27.5 % (v/v) ethylene glycol prior to freezing in liquid nitrogen. To obtain phase information, crystals were soaked in 1 mM KAu(CN)2 for 1 h prior to cryo-protection. Diffraction data of the AnkOTU crystals were collected at ESRF beamline ID23-2 to 2.23 Å (native) and 3.0 Å resolution at the peak-wavelength for Au. Phases were obtained by SIRAS, from an initial set of sites obtained with the SHELX/hkl2map suite . Site refinement was performed in SHARP , and subsequent density modification resulted in a high-quality map, which was interpreted by WarpNTrace and manually rebuilt in Coot . Refinement was performed using PHENIX , including simulated annealing and TLS B-factor refinement, resulting in the final statistics reported in . [...] 13C, 15N-labeled TRABID AnkUBD (aa 245-339) was expressed from a pOPIN-K vector in Rosetta2 pLacI cells. A 100 ml overnight culture grown in LB medium was pelleted and resuspended in modified K-MOPS minimal media , lacking nitrogen and carbon sources, and used to inoculate 3 L modified K-MOPS media supplemented with 13C glucose/15N ammonium chloride. Protein expression was induced after 16 h at 30˚C with 0.4 mM IPTG, and cells were harvested after a further 4 h. The protein was purified as for crystallography (see above) and concentrated to 10 mg ml-1. Unlabeled Ub (from bovine red blood cells, Sigma-Aldrich) was reconstituted in 50 mM Tris [pH 7.6] at 5 mM. 15N-labeled Ub was a kindly provided by Dr. Mark Allen (MRC LMB). Prior to data acquisition, samples were dialyzed against phosphate buffered saline (125 mM Tris [pH 7.2], 16.5 mM Na2HPO4, 8.5 mM NaH2PO4 × 2H2O) in 3 kDa cut-off Slide-A-Lyzer dialysis cassettes (Thermo Scientific). NMR experiments were acquired on Bruker DRX600MHz and AV2+ 700MHz spectrometers equipped with cryogenic triple resonance TCI probes and at a sample temperature of 298 K. All data were processed in Topspin 2.1 (Bruker, Karlsruhe) and analyzed in Sparky (Goddard & Kneller, UCSF). Standard triple resonance experiments (HNCACB, CBCA(CO)NH, HN(CA)CO and HNCO) were used to assign backbone resonances of the 13C, 15N double labeled TRABID AnkUBD, using a sample concentration of 180 μM. All observed correlations in the HSQC were assigned unambiguously, corresponding to 88 out of 95 non-Pro residues in the construct. Titration experiments and KD estimation are detailed in online. […]

Pipeline specifications

Software tools SHELX, HKL2MAP, Coot, PHENIX, Sparky
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens
Diseases Ovarian Neoplasms