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Protocol publication

[…] The coding sequence of CgDM9CP-1 was identified by searching the PMF against C. gigas genome database. The homology searches of amino acid sequence of CgDM9CP-1 were conducted with BLAST at the National Center for Biotechnology Information (NCBI). The protein domain was predicted with the simple modular architecture research tool (SMART) and conserved domain database (CDD) search service. Multiple alignment was performed with the Clustal X and the alignment show software Jalview. Conserved amino acid residues among DM9CPs were identified based on the sequence alignment and presented using WebLogo V3. [...] Phylogenetic tree was constructed as previously reported (). Briefly, the amino acid sequences of DM9CPs were searched against NCBI and the Joint Genome Institute. Multiple sequence alignments were generated using Clustal W with default parameters. The alignment was imported into the phylogenetic analysis program MEGA, and a maximum likelihood tree was generated. A circular phylogenetic tree was then constructed using the interactive tree of life server. [...] The structure was solved using the SHELXC/D/E pipeline with hkl2map as graphical user interface. Four sites were readily identified with SHELXD with a resolution cutoff of 2.6 Å resulting in CCall and CCweak of 46.0 and 28.5, respectively. Density modification in SHELXE was carried out for 60 cycles using the high resolution native data up to 1.1 Å resolution with a solvent content of 0.45. The initial model was auto built using BUCCANEER and completed with 144 of 144 residues built. The other structures were determined employing the Molecular Replacement Method using the native model DM9CP (PDB ID: 5MH0) as an ensemble in the program PHASER (). [...] The structures were refined initially using REFMAC5 () and PHENIX REFINE () for the final stages. Necessary model improvements as well as search for solvent molecules were performed using COOT () and “update water” in PHENIX REFINE. Anisotropic thermal displacement factors were refined at 1.3 Å or better resolution, otherwise using the TLS model. Glycerol was tentatively built in the model to allow for the corresponding electron density as well as d-mannose in the density of DM9CPm. […]

Pipeline specifications

Software tools SHELX, HKL2MAP, Buccaneer, REFMAC5, PHENIX, Coot
Organisms Drosophila melanogaster, Crassostrea gigas, Escherichia coli
Diseases Wiskott-Aldrich Syndrome
Chemicals Mannose