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Pipeline publication

[…] te and 0.1 M citric acid pH 5.0. Glycerol was added to a final concentration of 20% (v/v) as a cryo-protectant. Initial screening for diffraction was carried out using the Stanford Automated Mounting system (SAM) at the Stanford Synchrotron Radiation Lightsource (SSRL, Menlo Park, CA). The diffraction data were indexed in orthorhombic space group P212121., 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 ( […]

Pipeline specifications

Software tools PHENIX, HHPred, REFMAC5, ARP/wARP