Computational protocol: Structural Insights into the Recognition of Phosphopeptide by the FHA Domain of Kanadaptin

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[…] Native data were collected at wavelength 0.97932 Å at 100 K using a Pilatus 6M detector (DECTRIS) at SSRL beamline BL11-1. The data were processed by an automation script that runs XDS . The structure of the FHA domain of kanadaptin was determined by molecular replacement (MR). Initial MR “hybrid” model templates were created using the phenix.mr_model_preparation tool , which removes poorly aligned regions and trims side-chain atoms of non-conserved residues based on sequence alignments between the target sequence and top homologs in PDB calculated with the HHpred server . Multiple molecular replacement trials were carried out in parallel on a computer cluster with each job exploring different combinations of parameters (models, resolution, model completeness, and sequence similarity). Each job includes an MR step implemented in MOLREP , a rigid-body and restrained refinement step in REFMAC5 , followed by automatic model rebuilding in ARP/wARP . A MR solution was identified from a trial using the FHA domain of the Pml1p subunit of the yeast precursor mRNA retention and splicing complex (PDB ID 3els) as the search model. The resulting ARP/wARP model had an Rcryst of ∼20% and good completeness, and was confirmed by manual inspection of the corresponding density maps. Further model completion and refinement were performed manually with COOT and BUSTER . The refinement included TLS refinement with one TLS group per monomer and NCS restraints. Data and refinement statistics are summarized in . Analysis of the stereochemical quality of the model was accomplished using MolProbity . Molecular graphics were prepared with PyMOL (http://www.pymol.org/). Electrostatic potentials were calculated using the program Delphi . The structure factors and atomic coordinates are deposited in the RCSB Protein Data Bank (http://www.rcsb.org) with PDB codes 4h87. [...] Identification of domains and definition of domain boundaries were carried out using PFAM and HHpred . Secondary structure prediction was carried out using PSIPRED . Coiled-coil regions were predicted using MARCOIL and COILS/PCOILS . Homology modeling was performed with MODELLER and I-TASSER . Sequence alignments were calculated with CLUSTAL W2 , and rendered using TeXshade . […]

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