Computational protocol: Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery*

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[…] UBE2Z variants, UbcH5c, and BIRC6 were cloned into the pETNKI-His-3C-LIC-Kan vector following a ligation-independent cloning procedure (), with primers designed using ProteinCCD (). UBA6 was amplified using primers 5′-catgccatggaaggatccgagcctgtggcc-3′ and 5′-ccgctcgagatcagtgtcatgactgaagta-3′ and cloned into the pET24d vector (Novagen) using NcoI and XhoI sites. FAT10LRLR and UbCYCI mutants were generated using the QuikChange mutagenesis kit (Agilent). The ip-SUMO-FAT10 construct used for FAT10 expression was a gift from Marcus Groettrup. [...] Data were collected on beamlines ID23−1 at European Synchrotron Radiation Facility (France). UBE2Z crystals were in space group P21212 and diffracted to a resolution of 2.1 Å. Data reduction was done using XDS and XSCALE (, ). Molecular replacement trials were performed using PHASER () with UBE2D3 (PDB code 1X23) as the search model for UBE2Z. The initial model produced was improved upon using phenix.mr_rosetta (). Experimental phases were further acquired using crystals soaked in methylmercuric chloride. Iterative rounds of refinement were performed using either REFMAC () from the CCP4 suite () or BUSTER () and were interspersed with manual building in COOT (). Both the x-ray weight and B-factor restraint weight in REFMAC were optimized to reduce the Rwork/Rfree gap during refinement. Refined structures were optimized using local () and webserver () versions of PDB_REDO and were validated using the Molprobity server (). Structure figures were generated using PyMOL (). The UBE2Z structure has been deposited in the Protein Data Bank with accession code 5A4P. [...] Small angle x-ray scattering measurements on UBE2ZNter (residues 1–93), prepared in buffer 20 mm HEPES (pH 7.5), 150 mm NaCl, 10% (w/v) glycerol, and 1 mm TCEP, were performed on beamline BM29 at European Synchrotron Radiation Facility (France). The samples were thawed and centrifuged at high speed for 5 min before data acquisition. Samples were exposed to x-rays in a measuring cell cooled to 4 °C. Data were processed with Primus () from the ATSAS software package (). The quality of data at low angles was assessed from Guinier plots. […]

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