Crystallization and structure determination of the yeast Nup53 RRM domain
This protocol is extracted from research article:
Allosteric modulation of nucleoporin assemblies by intrinsically disordered regions
Sci Adv, Nov 27, 2019; DOI: 10.1126/sciadv.aax1836

Crystals of the Nup53 RRM domain from S. cerevisiae were grown at 4°C in sitting drops containing 1 μl of the protein (at 5 mg/ml) and 1 μl of a reservoir solution consisting of 20% (w/v) PEG-3350 (polyethylene glycol, molecular weight 800) and 0.2 M ammonium sulfate. The crystals belong to the space group C2221 and contain two RRM molecules in the asymmetric unit. For cryoprotection, crystals were stabilized in the mother liquor supplemented with 15% (v/v) glycerol and flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the Advanced Light Source, Lawrence Berkeley National Laboratory, beamline 8.2.1. The data were integrated and scaled using the HKL2000 package (49). The structure of the Nup53 homodimer was solved by molecular replacement (MR) using MOLREP (50) from the CCP4 (Collaborative Computational Project) program suite with the Nup53 RRM structure from Pichia guilliermondii [Protein Data Bank (PDB) accession code: 3P3D] as a search model. After MR, iterative cycles of manual rebuilding were carried out in COOT (51), and the final model was refined at a resolution of 1.75 Å using restrained refinement in PHENIX (52). The Fitmunk server (53) was used to identify improved side-chain rotamers. No electron density was observed for residues 287 to 304 due to predicted disorder. The stereochemical quality of the structural model was assessed with MolProbity (54). There were no residues in the disallowed region of the Ramachandran plot. Details of the data collection, phasing, and refinement statistics are provided in table S2. Structure-guided sequence alignment was generated with BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html). All figures containing the structure of the Nup53 RRM domain were generated with PYMOL (https://pymol.org/2/).

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