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A report on the crystal structure of a lectin isolated from Canavalia …


Biology Articles » Mycology » Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules » Methods

Methods
- Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

Crystallization and Data Collection

The purification of Canavalia gladiata lectin (CGL) was performed as discribed by Cecatto et al. [23] and Moreno et al. [24]. Crystals of the Canavalia gladiata lectin were grown by the hanging drop vapor diffusion method, drops contained 3 μL of protein solution and 3 μL of 0.1 M Tris-HCl, pH 8.5, with 2.0 M ammonium sulfate. Cryoprotected native CGL crystals diffracted to a resolution of 2.3 Å using a synchrotron radiation source and indexed, integrated and scaled at the same resolution. Low resolution data sets have been collected in capillar without cryoprotection at 2.9, 3.0 and 3.1 Å resolution in room temperature to confirm the presence of Abu. Native crystals were also soaked with a solution containing 1 mM α-methyl-mannoside. The complexed crystals diffracted to 2.3 Å. The data were indexed, integrated and scaled at 2.3 Å using MOSFLM [25] and SCALA [26]. Crystals belong to the orthorhombic space group C2221. The calculated Matthews coefficient indicated a tetramer in the asymmetric unit, and this protein assumes the same oligomerization in biological conditions.

Molecular Replacement and Refinement

The crystal structures CGL and CGL-αMM were determined by molecular replacement using the MolRep program [26]. Canavalia ensiformis lectin (ConA) structure without the complexed sugar and water molecules (PDB code 5CNA) was used as the search model [16]. The Abu coordinates were obtained by PRODRG program [27]. The initial structures were refined using REFMAC5 [28] and water molecules were added to the models using XtalView [29]. The crystal structures were deposited in the PDB [30] with accession code 1 WUV and 2D7F. The crystallographic statistics data are shown in Table 1. An omit map contoured at 3 σ to the aminobutyric acid was generated using CCP4 Omit program [28].

Mass Spectrometry Analysis

The presence of Abu in the molecule of native CGL was analyzed in an ESI-Q-ToF/MS/MS experiment. An amount of 1 mg of CGL was dissolved in 1 mL of a solution containing 50% acetonitrile and 5% TFA. The protein solution was diluted 100 × and injected in a nano-electrospray ionization source and analyzed in a Micromass™ quadrupole time-of-flight instrument with a resolution of 8,000 and accuracy of 10 ppm. The spectral data were processed using a Biolynx 4.0 program.


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