Computational protocol: Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1β (HNF1β) transcription factor is imported into the nucleus via a monopartite NLS sequence

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Protocol publication

[…] Diffraction quality crystals of the Importin-α1ΔIBB:HNF1βNLS complex were obtained by sitting drop vapour diffusion where 200 nl of 10% PEG 8 K, 0.09 M NPS, 20% ethylene glycol, 0.1 M Buffer 1 (Morpheus Screen, Molecular Dimensions) was mixed with 200 nl of Importin-α1ΔIBB:HNF1βNLS. The Importin-α1ΔIBB:HNF1βNLS complex was produced by mixing Importin-α1ΔIBB and HNF1βNLS peptide (Ac-TNKKMRRMRFK-NH2, Insight Biotechnology) at a 1:1.1 M ratio and a concentration of 6.7 mg/ml.Crystals were cryoprotected in the mother liquor supplemented with 20% glycerol and cooled by plunging into liquid nitrogen. X-ray diffraction data were collected on beamline I03 at the Diamond Light Source (Didcot, UK). Reflections were indexed and integrated using DIALS as implemented in Xia2 () and then scaled and merged in AIMLESS, ensuring a completeness of >98% in the outermost shell while maintaining CC1/2 > 0.3 (). The structure was solved by molecular replacement using Phaser with the structure of ΔIBB-mImportin-α1 complexed with a minor site small molecule inhibitor (PDB ID: 4U54 – ) to avoid introducing model bias into the NLS binding sites. Iterative cycles of rebuilding using COOT () and refinement using PHENIX () were used to generate the final model () that had an R-factor of 18.4% (R-free = 22.0%) and a MolProbity () score of 0.95 (100th percentile). […]

Pipeline specifications

Software tools Coot, PHENIX, MolProbity
Applications Small-angle scattering, Protein structure analysis
Diseases Ovarian Neoplasms