De novosulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse

Abstract

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.

High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

“…ratio for data set B was only 1.11, which is significantly below the empirically estimated threshold of 1.5 for successful S-SAD (Weiss et al, 2001;Mueller-Dieckmann et al, 2005). As previously noted by others (Sarma & Karplus, 2006;Lakomek et al, 2009), the R anom /R p.i.m. threshold may be adjusted to a lower limit, i.e.…”

“…threshold may be adjusted to a lower limit, i.e. 1.1, as found in the present study and as indicated previously in the S-SAD solution of the lysosomal mouse protein (Lakomek et al, 2009). Similarly, the SigAno parameter, or |Á ano |/(Á ano ), may also require adjustment.…”

The magic triangle goes MAD: experimental phasing with a bromine derivative

“…improved detectors, phasing with the weak anomalous signal from intrinsic scatterers has become a possible option, although high-quality data are required. These are more readily obtained when high symmetry enables a high data redundancy to be achieved, but low-symmetry examples have also proved successful (Lakomek et al, 2009).…”

S-SAD phasing study of death receptor 6 and its solution conformation revealed by SAXS

“…Two important factors contributed to the success of the lysozyme and obelin cases: (i) the development of the iterative single-wavelength anomalous scattering (ISAS) method coupled with solvent flattening (Wang, 1985) and (ii) advances in instrumentation and data-processing software which ensured the collection and processing of much more accurate anomalous diffraction data. Since then, many more de novo crystal structures have been solved using synchrotron X-rays (Gordon et al, 2001;Ramagopal et al, 2003;Brown et al, 2002;Weiss et al, 2004;Li et al, 2002;Madauss et al, 2004;Agarwal et al, 2006;Wagner et al, 2006;Lakomek et al, 2009;Goulet et al, 2010) or in-house Cu or Cr X-rays (Yang & Pflugrath, 2001;Debreczeni, Bunkó czi, Girmann et al, 2003;Debreczeni, Bunkó czi, Ma et al, 2003;Yang et al, 2003;Kitago et al, 2005;Nagem et al, 2005;Watanabe et al, 2005;Ren et al, 2005;Sarma & Karplus, 2006;Nan et al, 2009;Li et al, 2008).…”

“…Among the variables associated with data-collection strategies research papers involving longer wavelengths (Cianci et al, 2008;Ramagopal et al, 2003), the choice of the wavelength is one of the most important factors. To date, wavelengths for successful cases of de novo S-SAD phasing have mostly been within the range 1.54-2.50 Å (Lakomek et al, 2009;Weiss et al, 2001). The relationship between S-SAD signal and data quality using Xrays of wavelength beyond 2.5 Å has rarely been studied.…”