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 R_free 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 ų Da⁻¹.