Critical parameters for S-SAD phasing – real case experience

J. Stránský1,2, T. Kovaľ3, L. Østergaard4, J. Dušková1, T. Skálová1, J. Hašek1, P. Kolenko3, K. Fejfarová3, J. Dohnálek1,3

 

1Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague 4, the Czech Republic

2Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7, 11519 Prague 1, the Czech Republic

3Institute of Macromolecular Chemistry, Academy of the Sciences of the Czech Republic, Heyrovského nám. 2, 16206 Prague 6, the Czech Republic

4Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd Denmark

Jan.stransky@img.cas.cz

 

As X-ray diffraction technologies develop, de novo phasing of protein structures by single-wavelength anomalous dispersion by sulphur (S-SAD) is simplified and more common. However, anomalous differences in the sulphur atomic factors are in the order of errors of measurement, which makes careful intensity reading and data processing crucial. The new structure of a small 12 kDa protein with 4 sulphur atoms per molecule was phased with anomalous data up to 2.3 Å but the data did not enable a straightforward structure solution. Data processing was performed using XDS [1] and scaling using XSCALE. The sulphur substructure was determined by SHELXD [2] and phases were obtained by density modification and autotracing in SHELXE [2]. Both programmes strongly depend on input parameters and default values did not lead to the correct phases. Therefore a systematic search of optimal values of several parameters was used to find a solution. This method helped to confirm sulphur substructure and to differentiate the handedness of the solutions. Moreover, a script for comfortable conversion of SHELX outputs to MTZ format was developed, using programmes included in the CCP4 package [3]. The previously unsolvable protein structure was successfully resolved with the described procedure.

This work was supported by the Grant Agency of the Czech Technical University in Prague, (SGS13/219/OHK4/3T/14), the Czech Science Foundation (P302/11/0855), project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF.

1.       W. Kabsch, Acta Cryst., 2010, D66, 125-132

2.       G. M. Sheldrick, Acta Cryst., 2008, A64, 112-122

3.       M. D. Winn et al., Acta. Cryst., 2011, D67, 235-242