Computational protocol: Structural Basis for Par-4 Recognition by the SPRY Domain- and SOCS Box-Containing Proteins SPSB1, SPSB2, and SPSB4

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[…] hSPSB1 was buffered in 20 mM Hepes, pH 7.5, 100 mM NaCl, 10 mM l-arginine, 10 mM l-glutamate, and 10 mM DTT. hSPSB2 was buffered in 20 mM Hepes, pH 7.4, 150 mM NaCl, and 10 mM DTT. hSPSB4 was buffered in 50 mM Hepes, pH 7.5, 150 mM NaCl, and 5 mM DTT. Peptides were added to a final concentration of 2 mM. Crystallization was carried out using the sitting drop vapor diffusion method at 4 °C. The complex of hSPSB1/hPar-4 (I) was grown by mixing 75 nl of concentrated protein (4 mg/ml) with 75 nl of a well solution containing 0.2 M Li2SO4, 0.1 M 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol, pH 5.5, and 20% polyethylene glycol (PEG) 3350. The complex of hSPSB1/VASA (II) was grown by mixing 666 nl of the concentrated protein (4 mg/ml) with 333 nl of a well solution containing 0.2 M NaCl, 0.1 M 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol, pH 5.5, and 25% PEG 3350. The complex of hSPSB2/VASA (III) was grown by mixing 666 nl of the concentrated protein (9 mg/ml) with 333 nl of a well solution containing 0.22 M Na/KPO4, 11% ethylene glycol, and 22% PEG 3350. Apo-hSPSB4 (IV) was grown by mixing 100 nl of protein with 50 nl of a solution containing 14.4% PEG 10 K, 0.16 M Ca(ac)2, 20% glycerol, and 0.08 M cacodylate, pH 6.5. In all cases, crystals grew to diffracting quality within a few days and were cryo-protected using the well solution supplemented with 25% additional ethylene glycol and were flash frozen in liquid nitrogen.Data were collected at a Rigaku FRE Superbright equipped with an RAXIS IV detector at 1.5 Å (I), or at the Swiss Light Source beamline PX10 in the case of (II) (at 1.00001 Å) and (IV) (at 0.9537 Å), or at the Diamond Synchrotron beamline I03 in the case of (III) (at 0.9756 Å). Indexing and integration was carried out using MOSFLM and scaling was performed with SCALA for (I), (II), and (III) or XDS and SHELX for (IV). Initial phases for (I) and (IV) were calculated by molecular replacement with PHASER using 2FNJ as a starting model. Initial phases for (II) were calculated likewise using an ensemble comprising 2FNJ, 2V24, and 2IHS starting models. The models were completed manually in Coot and were refined with REFMAC5. The final model of (I) was used to obtain phases for (III) followed by manual build in Coot and refinement with REFMAC5. In all cases, thermal motions were analyzed using TLSMD and hydrogen atoms were included in late refinement cycles. Data collection and refinement statistics can be found in . […]

Pipeline specifications

Software tools iMosflm, CCP4, XDS, SHELX, Coot, REFMAC5
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens, Drosophila melanogaster