Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain α-neurotoxin from Naja atra venom

Atratoxin‐b, a short‐chain α‐neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three‐step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI–TOF mass spectrometry and SDS–PAGE. The protein is toxic to mice, with an in vitro LD50 of about 0.18 mg kg−1. Its N‐terminal amino‐acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other α‐­neurotoxins. The overall three‐dimensional structure of atratoxin‐b is very similar to that of the homologous erabutoxin‐a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and Rfree of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 × 0.1 × 0.15 mm over eight months using hanging‐drop vapour‐diffusion method. It gave a set of diffraction data to 1.56 Å resolution using X‐­rays of wavelength 1.1516 Å generated by the X‐ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P41212, with unit‐cell parameters a = 49.28, c = 44.80 Å, corresponding to one molecule per asymmetric unit and a volume‐to‐mass ratio of 1.96 Å3 Da−1.