Computational protocol: Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

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Protocol publication

[…] We used a blastp search for the amino acid sequence of IPO [GenBank: BAA14024.1] against the algorithm of the National Center for Biotechnology Information (NCBI) protein databank database for searching structural templates. The amino acid sequence of Calystegia sepium agglutinin (Calsepa), a JRL (PDB: 1OUW), showed 53% sequence identity to that of IPO. The monomeric structure of Calsepa was further used in a search to determine the structure of apo IPO by molecular replacement with use of the program CNS . After cross-rotation and translation of molecular replacement, 4 values were obtained. Initial rigid body refinement for the 4 monomeric structures gave a 48.8% R-factor. Clear continuous electron density could be observed after calculation of Fourier maps, and the 5th molecule of apo IPO was further built accordingly. Because of different space groups for the structures of the IPO–Me-Man, IPO–Me-Glc and IPO–Me-Gal complexes, the resolved monomeric apo IPO was used as a search template in the following molecular replacement method. The solutions of cross-rotation and translation could be obtained with 2 molecules for the IPO–Me-Man complex, 4 molecules for IPO–Me-Glc and 4 molecules for IPO–Me-Gal. Those solutions were further applied to initial rigid body refinement, and reasonable values were obtained (e.g., 36.7% R-factor for IPO–Me-Man, 35.4% for IPO–Me-Glc, and 32.9% for IPO–Me-Gal).Manual model rebuilding involved use of Coot , alternating refinement by the CNS program, with 5% or 10% of the observed reflections randomly selected and set aside for calculation of the Rfree value. The final refined statistics are in . For the protein interface of the tetrameric form, IPO–Me-Glc was used as a representative for analysis by the web service PDBe PISA . All molecular representations were prepared with use of DeepView and PyMOL . The coordinates of monomers of apo IPO (e.g., chain A) were subjected to the web service Matras for structure comparison . […]

Pipeline specifications

Software tools BLASTP, CNS, Coot, Swiss-PdbViewer, PyMOL, MATRAS
Databases PDBe
Application Protein structure analysis
Organisms Ipomoea batatas
Chemicals Carbohydrates