Purification of hemachatoxin from the venom of <i>H. haemachatus</i>.

<p>(<b>A</b>) Size-exclusion chromatogram of the crude venom. The proteins were eluted using 50 mM Tris-HCl, pH 7.4 and monitored at 280 nm. The fractions of peak 3 (<i>black horizontal bar</i>) were pooled and sub-fractionated on RP-HPLC. (<b>B</b>) RP-HPLC chromatogram of peak 3 using a linear gradient of 28–50% solvent B. The elution was monitored at 215 nm. The <i>black arrow</i> indicates the elution of hemachatoxin. (<b>C</b>) The re-purification of hemachatoxin on a shallow gradient of 35–45% solvent B. The elution was monitored at 215 nm. (<b>D</b>) The ESI-MS profile of hemachatoxin showing the three peaks of mass/charge (m/z) ratio ranging from +4 to +6 charges. The mass of hemachatoxin was determined to be 6835.68±0.94 Da.</p

Crystallographic data and refinement statistics.

<p>Statistics from the current model.</p>a<p>R_sym = Σ|I_i−<i>|/Σ|I_i| where I_i is the intensity of the i^th measurement, and <i> is the mean intensity for that reflection.</i></i></p><i><i>b<p>R_work = Σ| F_obs−F_calc|/Σ|F_obs| where F_calc and F_obs are the calculated and observed structure factor amplitudes, respectively.</p>c<p>R_free = as for R_work, but for 10.0% of the total reflections chosen at random and omitted from refinement.</p>*<p>Values in the parenthesis are the highest resolution bin values.</p></i></i

Comparison of hemachatoxin with other three-finger toxins.

<p>(<b>A</b>) Structure based sequence alignment of hemachatoxin and its homologs, cardiotoxin 3 (1H0J), cytotoxin 3 (1XT3), cardiotoxin A3 (2BHI), cardiotoxin VI (1UG4) and cardiotoxin V (1KXI), (all from <i>Naja atra</i>), cardiotoxin V_II4 (1CDT) from <i>Naja mossambica</i> and toxin-γ (1TGX) (a cardiotoxin from <i>Naja nigricollis</i>). This figure was generated using the programs ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Larkin1" target="_blank">[78]</a> and ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Gouet1" target="_blank">[79]</a>. (<b>B</b>) Comparison of hemachatoxin with its structural homologs. Hemachatoxin (brown), cardiotoxin 3 [1H0J] (cyan), cytotoxin 3 [1XT3] (black), carditotoxin A3 [2BHI] (blue), cardiotoxin VI [1UG4] (red), cardiototoxin V [1KXI] (pink), cardiotoxin V_II4 [1CDT] (green) and toxin-γ [1TGX] (yellow).</p

Multiple sequence alignment of hemachatoxin with cardiotoxins/cytotoxins (A) and other three-finger toxins (B).

<p>Toxin names, species and accession numbers are shown. Conserved residues in all the sequences are highlighted in black. The type of cardiotoxin based on the conserved Pro31 is highlighted in grey. Disulfide linkages and loop regions are also shown. The sequence identity (in percentage) of each protein with hemachatoxin is shown at the end of each sequence.</p

Structure of hemachatoxin.

<p>(<b>A</b>) Ribbon representation of the hemachatoxin monomer. Cysteine bonds are shown in <i>yellow.</i> β-strands, N- and C- terminals are labeled. (<b>B</b>) Electron density map<b>.</b> A sample final <i>2Fo-Fc</i> map of hemachatoxin shows the region from Tyr23 to Lys29. The map is contoured at a level of 1σ. (<b>C</b>) The electrostatic surface potential of hemachatoxin is shown in the same orientation as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone-0048112-g003" target="_blank">Figure 3A</a>. Blue indicates positive potential and red indicates negative potential in units kT/e. All the structure related figures of this paper were prepared using the program PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Delano1" target="_blank">[77]</a>.</p