Section 16.1.8.3. Data resolution and completeness

The importance of the presence of several atoms heavier than oxygen for increasing the chance of obtaining a solution by SnB at resolutions less than 1.2 Å was noticed for truncated data from vancomycin and the 289-atom structure of conotoxin EpI (Weeks & Miller, 1999b). The results of SHELXD application to hirustasin are consistent with this (Usón et al., 1999). The 55-amino-acid protein hirustasin could be solved by SHELXD using either 1.2 Å low-temperature data or 1.4 Å room-temperature data; however, as shown in Fig. 16.1.8.2(a), the mean phase error (MPE) is significantly better for the 1.2 Å data over the whole resolution range. The MPE is determined primarily by the data-to-parameter ratio, which is reflected in the smaller number of reliable triplet invariants at lower resolution. Although small-molecule interpretation based on peak positions worked well for the 1.2 Å solution (overall ), standard protein chain tracing was required for the 1.4 Å solution (overall ). As is clear from the corresponding electron-density map (Fig. 16.1.8.2b), the Shake-and-Bake procedure produces easily interpreted protein density even when bonded atoms are barely resolved from each other. The hirustasin structure was also determined with SHELXD using 1.55 Å truncated data, and this endeavour currently holds the record for the lowest-resolution successful application of Shake-and-Bake.

Figure 16.1.8.2| top | pdf | (a) Mean phase error as a function of resolution for the two independent ab initio SHELXD solutions of the previously unsolved protein hirustasin. Either the 1.2 Å or the 1.4 Å native data set led to solution of the structure. (b) Part of the hirustasin molecule from the 1.4 Å room-temperature data after one round of B-value refinement with fixed coordinates.

The relative effects of accuracy, completeness and resolution on Shake-and-Bake success rates using SnB for three large P1 structures were studied by computing error-free data using the known atomic coordinates. The results of these studies, presented in Table 16.1.8.3, show that experimental error contributed nothing of consequence to the low success rates for vancomycin and lysozyme. However, completing the vancomycin data up to the maximum measured resolution of 0.97 Å resulted in a substantial increase in success rate which was further improved to an astounding success rate of 80% when the data were expanded to 0.85 Å.

On account of overload problems, the experimental vancomycin data did not include any data at 10 Å resolution or lower. A total of 4000 reflections were phased in the dual-space loop in the process of solving this structure with the experimental data. Some of these data were then replaced with the largest error-free magnitudes chosen from the missing reflections at several different resolution limits. The results in Table 16.1.8.4 show a tenfold increase in success rate when only 200 of the largest missing magnitudes were supplied, and it made no difference whether these reflections had a maximum resolution of 2.8 Å or were chosen randomly from the whole 0.97 Å sphere. The moral of this story is that, when collecting data for Shake-and-Bake, it pays to take a second pass using a shorter exposure to fill-in the low-resolution data.