olda br Structure modeling and refinement The structural pha

Structure modeling and refinement The structural phase information was initially obtained by the molecular replacement method, by using the software Phaser in CCP4 [5,6]. The published structures of APRIL (PDB accession code: 1Q5X) and BAFF (PDB code: 1KD7) were used as search models. About 3% of the measured reflections were excluded for the calculation of the free R-factor in order to cross-validate the correctness of the final model. Subsequent model building was done in multiple rounds using software COOT. Refinement was performed using the REFMAC5 software with bulk solvent correction and TLS parameterization in the CCP4 package [6–9]. The water model was built with the “Find waters” algorithm of COOT by putting water molecules in peaks of the Fo–Fc map contoured at 3.0 sigma, followed by refinement with REFMAC5 and checking all waters with the validation tool of COOT. The occupancy of side chains, which were in negative peaks in the Fo–Fc map (contoured at −3.0 sigma), were set to zero. The model was further subjected to the refinement using software BUSTER [10]. The final refinement residual factors, Rwork and Rfree, are 17.9% and 23.4%, respectively. The r.m.s. deviations for bond length and bond angle are 0.01Å and 1.22°, respectively. The Ramachandran Plot of the final model shows 95% of all residues in the favored region, and 0.5% in the outliers region [11] and is in agreement with the main-chain conformational tendencies shown in an earlier study [12].
The deposited data The structure contains two APRIL–BAFF–BAFF heterotrimers in each asymmetry unit. A total of 6997 atoms (6650 from protein, 339 from water and 8 from a TRIS buffer molecule) were included in the final model. Table 5 is the list of amino olda residues in the final model and their corresponding amino acids in the natural mature protein [13]. The atomic coordinates and structure factors have been deposited into the Protein Data Bank (http://www.rcsb.org) with the accession code 4ZCH.
Acknowledgment
Specifications table
Value of the data
Data EGCG inhibited the MMP-2 activity in cell-free assays using HT1080 conditioned medium or the purified active enzyme (Fig. 1). Pretreatment with 10mM EDTA enhanced EGCG-mediated inhibition of MMP-2 (Fig. 2). Chelating agents alone did not affect MMP-2 activity (Fig. 3).
Experimental design, materials and methods
Acknowledgements We thank Dr. Vikash Kumar Dubey and his lab members for the technical help and Mohan C.M. for helping in manuscript preparation. GD is the recipient of the Shyama Prasad Mukherjee Fellowship awarded by Council of Scientific and Industrial Research, Government of India and acknowledges the financial support. AML acknowledges research funding from the Department of Science and Technology, Government of India (Letter no. SR/FT/LS-028/2009) and the Indian Council of Medical Research, Government of India (Letter no. 5/7/773/12-RCH).
Specifications Table
Data, experimental design, materials and methods
Conflict of interest
Acknowledgments This work was supported by Grants-in-Aid both for Scientific Research from Japanese MEXT and MHLW. This work was also supported by Grants from Scientific Research on Innovative Areas (the Glial Assembly area and the Comprehensive Brain Science Network area), the Astellas Science Foundation, and the Takeda Science Foundation.
Data Two coding sequences were obtained for IGF-IRs in gilthead sea bream and deposited in GenBank as IGF-IRa (Accession number: KT156846) and IGF-IRb (Accession number: KT156847). The sequence analysis revealed that both receptors are composed by one α and one β subunits connected by the characteristic tetrabasic cleavage site and, are organized into several major domains characteristic of this family of receptors (Fig. 1). The phylogenetic analysis showed that the tree divides first into two distinct branches, one including the mammalian, avian, reptilian and amphibian sequences and, a second one containing only fish orthologues, which separates again creating two clusters containing one paralogue each (Fig. 2). Furthermore, the two gilthead sea bream IGF-IRs shared a protein identity of 69% and had values of comparison with other teleosts that ranged among 62–86% (Table 1). Additionally, a semi-quantitative PCR analysis of both receptors showed that IGF-IRa was expressed equally in all tissues analyzed, whereas IGF-IRb presented differential expression between tissues, being more expressed in brain, spleen, stomach, heart and gill, and to a lower extent in the remaining tissues (Fig. 3).