Computational protocol: Laminarinase from Flavobacterium sp. reveals the structural basis of thermostability and substrate specificity

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Protocol publication

[…] The initial phase of ULam111SeMet was obtained using single anomalous dispersion (SAD). After selenium atom search and phase calculations with PHENIX AutoSol Wizard in the PHENIX program suite, model building was automatically carried out with PHENIX AutoBuild Wizard. Manual rebuilding and refinement of ULam111SeMet were performed with COOT and PHENIX.REFINE. The structure of native ULam111 was determined by the molecular replacement method using the MOLREP program, with the ULam111SeMet structure as the initial model. Manual rebuilding and refinement of native ULam111 was performed with COOT and PHENIX.REFINE, respectively. The geometry of the final structure was evaluated with the program Rampage. The coordinates of ULam111 have been deposited into the Protein Data Bank (PDB) with the accession number (5WUT). [...] Structural analysis was carried out using a set of programs: Dali was used for the search of similar structures from the database, DaliLite was used for the superposition of molecules, ESpript was used for the preparation of alignment figures, and Pymol (http://pymol.sourceforge.net/) for the depiction of structures.We failed to obtain cocrystals of ULam111 and laminarin. Two types of tetrasaccharides (β-1,3-linkages and β-1,3-1,4-linkages, respectively) were modeled on the ULam111 structure based on the superposition of β-1,3-glucanase ZgLamA from Zobellia galactanivorans (PDB code: 4BOW; sequence identity, 34%; preferable β-1,3-linkages) and β-1,3-1,4-glucanase H(A16-M) (PDB code: 1U0A; sequence identity, 17%; preferable β-1,3-1,4-linkages). Gluconolactone was modeled on the superposition of TmLam from Thermotoga maritima (PDB code: 3AZX). […]

Pipeline specifications

Software tools PHENIX, Coot, Molrep, DaliLite, ESPript, PyMOL
Application Protein structure analysis
Chemicals Calcium, Hydrogen