Computational protocol: Structural studies of the N-terminal fragments of the WW domain: Insights into co-translational folding of a beta-sheet protein

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[…] The proteins of WWX-MBP were concentrated to 20 mg/mL in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 10 mM maltose. All crystallization attempts were made using the vapor-diffusion method at 293 K. Crystals were obtained in a solution that was a 1:1 mixture of protein solution and reservoir solution. The reservoir conditions differed for the individual variants as follows: 1.8 M ammonium citrate was used for Form I of WW11-MBP, 2.2 M DL-malic acid for Form II of WW11-MBP, 2.1 M DL-malic acid for WW17-MBP, 1.6 M ammonium citrate for WW19-MBP, and 1.6 M ammonium citrate for WW35-MBP. The crystals were flash cooled in a cold stream of nitrogen after soaking in the cryoprotectant solutions consisting of reservoir solution with 20% (w/v) glycerol. The X-ray diffraction data were collected with synchrotron X-rays from BL41XU (1.0000 Å, 100 K), BL44XU (0.9000 Å, 100 K) of SPring-8 (Harima, Japan), or with CuKα X-rays from an in-house rotating anode X-ray generator MicroMax-007 (1.5418 Å, 100 K) (Rigaku). Diffraction data sets were processed and scaled using the HKL2000 software package. Form I of WW11-MBP is in orthorhombic crystal form and belongs to the space group C2221 with unit-cell parameters a = 97.5 Å, b = 126.1 Å, c = 173.5 Å. There are two molecules in an asymmetric unit. Form II of WW11-MBP is in tetragonal crystal form and belongs to the space group P41212 with unit-cell parameters a = 115.6 Å, b = 115.6 Å, c = 55.6 Å. There is one molecule in an asymmetric unit. The crystals of WW17-MBP and WW19-MBP belong to space group P212121, with unit-cell parameters a = 51.2 Å, b = 73.0 Å, c = 100.8 Å and a = 48.3 Å, b = 57.7 Å, c = 124.2 Å, respectively. The crystals of WW17-MBP and WW19-MBP contain one molecule in an asymmetric unit. The crystals of WW35-MBP belong to space group P21, with unit-cell parameters a = 84.5 Å, b = 120.4 Å, c = 110.7 Å, β = 97.8°. There are four molecules in the asymmetric unit. All structures were solved by the molecular replacement method using MBP (PDBID: 1ANF) as a search model with the program Phaser. The phenix.autobuild program was employed for the autotracing. The output structures were manually improved with the Coot program. The structures were refined using the phenix.refine. The validity of the refined structures was checked with the program MolProbity. The crystallographic and refinement statistics are listed in . The superimpositions were performed with the program LSQKAB. The secondary structure was assigned using the program DSSP. All figures for the molecular models were prepared using the program PyMOL. […]

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