MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it X-ray crystallography and NMR studies of domain-swapped canecystatin-1. X-ray crystallography and NMR studies of domain-swapped canecystatin-1. FEBS J. (1742-464X) 2013 Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction experimental particp experimental participant identification experimental particp Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. FEBS J. (1742-464X) 2013 X-ray crystallography and NMR studies of domain-swapped canecystatin-1. X-ray crystallography and NMR studies of domain-swapped canecystatin-1. FEBS J. (1742-464X) 2013 Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. FEBS J. (1742-464X) 2013 X-ray crystallography and NMR studies of domain-swapped canecystatin-1. X-ray crystallography and NMR studies of domain-swapped canecystatin-1. FEBS J. (1742-464X) 2013 Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr experimental particp experimental participant identification experimental particp Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. FEBS J. (1742-464X) 2013 X-ray crystallography and NMR studies of domain-swapped canecystatin-1. X-ray crystallography and NMR studies of domain-swapped canecystatin-1. FEBS J. (1742-464X) 2013 Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. in vitro In vitro dls dynamic light scattering dls experimental particp experimental participant identification experimental particp Valadares NF., de Oliveira-Silva R., Cavini IA., Marques Ide A., Pereira HD., Soares-Costa A., Henrique-Silva F., Kalbitzer HR., Munte CE., Garratt RC. FEBS J. (1742-464X) 2013 q7y0q9_sacof Cysteine proteinase inhibitor protein protein sugarcane Saccharum officinarum MAEAHNGRRVGMVGDVRDAPAGHENDLEAIELARFAVAEHNSKTNAMLEFERLVKVRHQVVAGTMHHFTVQVKEAGGGKKLYEAKVWEKVWENFKQLQSFQPVGDA mint 1D86CBC56F7960F5 q7y0q9 2 Canecystatin-1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Stoichiometry: 2.0 direct interaction direct interaction mint f1 sf1 The expected molecular mass of His-tag free canecystatin-1, based on its amino acid sequence, is 11.9 KDa. Therefore, it is likely that the third (poorly resolved) peak observed on gel filtration, with an estimated molecular mass of 14.5 KDa, corresponds to the monomeric species. On the other hand, the predominant second peak, eluting with an estimated mass of 33.4 KDa, is probably a homodimer. This is consistent with the results of dynamic light scattering which yielded a single peak corresponding to a mass of 38.5 ± 11.9 KDa (Supplementary Figure 2). The fact that this dimer is expected to be domain swapped in solution (see below)and therefore elongated in shape, probably explains the overestimation of its molecular mass by both techniques. The first peak (59.3 KDa) is most likely due to a small population of tetramers. q7y0q9-1 2 Canecystatin-1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Stoichiometry: 4.0 direct interaction direct interaction mint f1 sf1 The expected molecular mass of His-tag free canecystatin-1, based on its amino acid sequence, is 11.9 KDa. Therefore, it is likely that the third (poorly resolved) peak observed on gel filtration, with an estimated molecular mass of 14.5 KDa, corresponds to the monomeric species. On the other hand, the predominant second peak, eluting with an estimated mass of 33.4 KDa, is probably a homodimer. This is consistent with the results of dynamic light scattering which yielded a single peak corresponding to a mass of 38.5 ± 11.9 KDa (Supplementary Figure 2). The fact that this dimer is expected to be domain swapped in solution (see below)and therefore elongated in shape, probably explains the overestimation of its molecular mass by both techniques. The first peak (59.3 KDa) is most likely due to a small population of tetramers. q7y0q9-1 1 Canecystatin-1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Stoichiometry: 2.0 direct interaction direct interaction mint The coordinates and structure factors were deposited in the Protein Data Bank under accession codes 3UL5 and 3UL6 The crystal structures of canecystatin-1 in both orthorhombic and hexagonal space groups were readily solved by the molecular replacement method. After the first refinement cycle, in both structures the electron densities of the first inhibitory hairpin loop (residues Val60 to Gly63, located between β-strands 2 and 3) unambiguously showed that the individual subunits were present in the form of domain swapped dimers, as can be seen from the omit map shown in Supplementary Figure 3. The asymmetric unit of the orthorhombic structure contains two domain swapped dimers, while that of the hexagonal structure contains only one. These dimers present a good overall superposition to one another with RMS deviations varying between 0.8 and 2.1 Å, and their overall fold is similar to other cystatins, consisting of a four-stranded antiparallel β-sheet (composed of three strands from one subunit and one from the other) curved around an α-helix (Fig. 2). t1 sf3 sf5a f2 f7 q7y0q9-1 3 Canecystatin-1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Stoichiometry: 2.0 direct interaction direct interaction mint f3 f4 f5 q7y0q9-1 4 Canecystatin-1 5 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 2.0 direct interaction direct interaction sf2 mint