Nature research paper: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel
wild-type strain SMD1168 was obtained from Life Technology. Transformations were performed by electroporation, following the manufacturer’s instructions. Transformed yeast cells were grown on Zeocin-containing YPDS plates at 30 °C for 3 days. A single colony was picked to inoculate a starting culture, which was incubated at 30 °C overnight. A large culture was then inoculated by diluting the starter culture 1:100 into buffered minimal glycerol medium.
For nanodisc reconstitution, a lipid stock was first prepared. The stock contained 10 mM each of 1,2-dioleoyl-sn-glycero-3-phosphocholine , 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleoyl-sn-glycero-3-phospho-. Protein purified in digitonin was incorporated into nanodisc using a 1:240:5 molar ratio of protein:lipid:membrane-scaffold protein 1D1 . This mixture was incubated at 4 °C for 2 h with gentle agitation.
To assemble a Fab–ligase complex, purified ligase complex in digitonin was incubated with Fab at a 1:1.5 molar ratio on ice for 1 h. The Fab–ligase complex was concentrated and loaded on a Superose 6 3.2/300 Increase size-exclusion column in buffer C . Peak fractions were pooled and concentrated to approximately 7 mg mlE. colifor 30 min at 4 °C, and the filtered supernatant was applied to a glutathione resin .
For purification of the Pex12 ring finger domain used for crystallization, GST-3C–Pex12 RF bound to the glutathione resin was incubated with PreScission protease at 4 °C overnight to cleave off the GST tag. The flow-through fraction was collected and loaded onto a Mono Q ion-exchange column . The protein was eluted with a salt gradient and peak fractions were pooled.
The Pex12 ring finger domain was crystallized by the hanging-drop vapour diffusion method by mixing 0.2 μl of the protein at 6 mg mlwith 0.2 μl of the 100 μl reservoir solution containing 0.1 M bis-Tris propane, pH 8.5, 0.2 M sodium fluoride and 20% PEG3350. Crystals were cryo-protected by gradually increasing the glycerol concentration in the drop by repeated additions of well solution supplemented with 30% glycerol.
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