Section 26.1.3.7. The crystal-type problem

This discovery was particularly galling because, although at the time variations in diffraction patterns had been reported for some protein crystals, we had failed to notice that this phenomenon had been mentioned years earlier in a study of lysozyme (Corey et al., 1952).

A preliminary analysis of the differences in the diffraction patterns suggested two important characteristics:

The two diffraction patterns were characterized operationally by specific patterns of intensities in the 3–4 Å resolution range (where the differences appeared to be maximal), and particularly by a few adjacent pairs of reflections whose relative intensities interchanged in the two types. The principal diagnostic reflections were and . In data associated with crystal type I, , while in crystal type II, . The L rows of reflections from the two types of crystals had the structure amplitudes shown in Table 26.1.3.1.

Table 26.1.3.1| top | pdf | Structure amplitudes of the reflections from crystal types I and II Crystal type Reflection I 29.2 21.8 10.6 18.2 23.1 17.0 II 36.9 25.7 19.7 27.7 26.4 36.8

In order to stand any chance of successfully calculating the lysozyme map by isomorphous replacement, we had to sort all the data so far collected, both native and derivative, into the two types and then recollect `rogue' data sets in order to assemble complete data sets of one particular type. The alternative was to recollect the whole data on a sounder basis, which we were loath to do, especially as other teams seemed likely to be well advanced in their solution of the lysozyme structure.

It appeared that the bulk of the data that had already been collected using the diffractometer was what we had called type II. We also observed that nearly all the photographic data collected in the heavy-atom proving stage was of type I. This observation was of great importance because it gave us a sound basis for defining the differences between the type I and II diffraction patterns, and it also provided a vital clue in identifying from their shapes the crystals that gave the two types of diffraction pattern. This was very important to us in the selection of crystals to replace the rogue data sets. The crystals that gave the best results for the photographic work tended to be relatively small and flattened along the tetragonal fourfold axis direction, while those that gave the best diffractometer data were the larger isometric crystals, which were more extended along the crystal fourfold axis (Fig. 26.1.2.1). These crystals could be definitely associated with the type I and type II diffraction patterns, respectively. Batches of lysozyme crystals grown according to the procedures defined earlier usually contained both types of crystal. This suggested that the two crystal forms might have originated when the pH of crystallization was on the borderline between two crystal forms of lysozyme. This hypothesis was supported by the observation that crystals grown at a somewhat higher pH had diffraction patterns closely similar if not identical to type I.

The more thorough analysis of the differences between the diffraction patterns for types I and II that these findings permitted showed that the differences in the intensities of equivalent reflections were resolution-dependent. They were very small in the 6 Å region (which probably accounts for the differences not being observed earlier), increased to a maximum at the position of the normal 4 Å peak in the protein diffraction pattern, and fell off at higher resolutions. This pattern is consistent with a lack of isomorphism between the two types of crystal of the kind that may be caused by a slightly different orientation of the lysozyme molecule in the unit cells of the two crystals (Crick & Magdoff, 1956). Such effects may be brought about, for example, by slightly different charge distributions in the protein molecules, rather than by the presence of additional diffracting material in one crystal type or the other. [This conclusion was later confirmed by a detailed analysis of the two structures by Helen Handoll (1985).]

These observations suggested it was safe to go ahead with trying to determine the structure of lysozyme in either type of crystal, and the decision to proceed with the type II crystals was solely on the basis that the bulk of the data already collected were of this type. Knowing the characteristic pairs of reflections that distinguished the two crystal types, we found it relatively straightforward to ascribe each data set to a particular crystal type, even for the heavy-atom derivatives, because the type differences were much larger than the heavy-atom changes at approximately 4 Å resolution. All triplet levels of data that belonged to the type I diffraction pattern were extracted from the total data sets and replaced by equivalent data sets recollected from confirmed type II crystals. This process, whose success was carefully tested and confirmed during the data processing and reduction stage, resulted in consistent data sets at 2 Å resolution for the native lysozyme and all three isomorphous derivatives that were derived from type II crystals.