Section 26.1.3.3. The second low-resolution map at 6 Å

Our purpose in calculating a new electron-density map at 6 Å was fourfold. First to ascertain whether the procedures used to identify the five derivatives thought to be satisfactory at this level of resolution had worked satisfactorily. Second, to judge the quality of the measurements made by the triple-counter diffractometer. Third, to explore the effects of the modified method of applying absorption corrections to the intensities that are described below, although these were not expected to have a very great effect at low resolution. Fourth, to examine the effectiveness of the new procedure for incorporating anomalous-scattering information in the phase determination, which is also described below.

Comparison of the two sets of structure amplitudes gave a conventional R value of 0.075, which is not particularly good – perhaps because of the comparatively large background values associated with these low-angle measurements. However, the mean figure-of-merit obtained in the new phase calculations was 0.97 as compared with the 0.86 obtained originally. The root-mean-square difference in electron density between the two maps was 0.012 e Å⁻³, from which it may be judged that the two maps were very similar. Nevertheless, the outline of the molecule was certainly clearer in the new map, and within the molecule there was improved continuity, suggesting the course of a folded polypeptide chain, and there were a number of stronger rod-like features suggestive of α-helices. Two of these were prominent, running upwards from right to left, in the view of the new model shown in Fig. 26.1.3.3.

Figure 26.1.3.3| top | pdf | Solid model of the electron density greater than about 0.5 e Å−3 in the second study of lysozyme at 6 Å resolution. This view of the model is equivalent to a view of the original model seen horizontally from the right of Fig. 26.1.2.13(c). (a) The new model has a marked cleft running roughly vertically down the other side of the model, corresponding to the one that can be seen in Fig. 26.1.2.13(c). (b) The cleft was shown to bind inhibitor molecules. The black density is that observed for the lysozyme–GlcNAc complex at 6 Å resolution.

The result was very encouraging, and we therefore went ahead immediately with data collection at 2 Å resolution, using essentially the same methods. At the same time, we began to plan low-resolution studies of inhibitor binding to lysozyme, from which we hoped to derive information about the nature of the enzyme–substrate complex.