Section 26.1.2.8. The electron-density map of lysozyme at 6 Å resolution

The electron-density distribution was calculated on the MERCURY computer, by means of a program written by Owen Mills that was in general use at the time, with structure amplitudes weighted by the figures of merit so as to give the `best' Fourier (Blow & Crick, 1959). The map was contoured by hand and plotted on clear plastic (Perspex; Plexiglass in the USA) sheets (Fig. 26.1.2.12).

Figure 26.1.2.12| top | pdf | Electron-density distribution in lysozyme at 6 Å resolution viewed parallel to the c axis. The horizontal and vertical lines represent the twofold rotation axes and intersect the twofold screw axis, upper right of centre. The fourfold screw axis is at the lower left of centre. The contour interval is 0.07 e Å−3, the lowest heavy contour being at 0.6 e Å−3. The absolute scale is approximate. Reproduced with permission from Nature (Blake et al., 1962). Copyright (1962) Macmillan Magazines Limited.

The first objective in studying this map was to determine the boundary of a single molecule. Comparison of the unit cells of various crystal forms of lysozyme (Steinrauf, 1959) suggested that in tetragonal lysozyme, the molecule occupies the full length of the c axis and, on average, one-eighth of the ab plane. On this basis, the map of Fig. 26.1.2.12 includes the whole of the c axis and a sufficient area of each section to ensure that one whole molecule is included in addition to parts of neighbouring molecules. Only contours indicating where the electron density is greater than average are included.

Some featureless regions of average electron density (0.4 e Å⁻³) were immediately apparent. The most marked of these was around the twofold screw axis parallel to c. This axis is intersected at intervals of 9.5 Å by twofold rotation axes, and packing considerations, therefore, made it impossible for substantial parts of the molecule to penetrate into the neighbourhood. Similarly, the immediate vicinity of the fourfold screw axis was also without significant features and was clearly a region of intermolecular space. The twofold rotation axes also helped to determine the boundary, particularly where relatively high density approached or intersected them, as it did in two places. It was clear that such regions must represent close contacts or bridges between adjacent molecules.

Following the example of the haemoglobin study (Perutz et al., 1960), we decided at this stage to make a balsa-wood model of the electron density to help visualize the molecule. Instead of producing a stack of sections through the molecule, however, CCFB devised a way of shaping the sections to make a smooth model of the volume occupied by electron density greater than about 0.53 e Å⁻³. The result is shown in Fig. 26.1.2.13.

Figure 26.1.2.13| top | pdf | Views of a 6 Å resolution model of the regions in which the electron density exceeds about 0.53 e Å−3. The vertical rods indicate the twofold and fourfold screw axes parallel to c. The thinner horizontal rods are twofold rotation axes. At two places, shown hatched, rotation axes pass through continuous regions of density. The white regions alone make up the most compact asymmetric unit of the structure. In part (b), the grey section (B) is alternative to the region (A), to which it is related by the twofold axis T. In part (a), the grey section (D) is alternative to the white (C), related by the fourfold screw axis. The grey piece (F) in part (c) is alternative to the white (E), to which it is related by a unit-cell translation along c. The scale is indicated by the framework of symmetry elements, adjacent parallel twofold axes being 18.95 Å apart. Reproduced with permission from Nature (Blake et al., 1962). Copyright (1962) Macmillan Magazines Limited.

One asymmetric unit is shown in white, with additional pieces in grey to show alternative shapes. The white pieces together make up the most compact asymmetric unit, which is roughly ellipsoidal in shape with axes 52 × 32 × 26 Å. Later work showed that this asymmetric unit represented a single molecule but, at this stage, we were scrupulous in detailing the alternative interpretations. There is a region of low density that divides the model roughly into two halves, and although we speculated about this we refrained from making any suggestions about its possible significance in our description of the structure (Blake et al., 1962). Instead, we noted that the two halves of the model could be assembled differently, following the crystal symmetry, so as to form a dumb-bell shaped molecule connected at PP′ (Fig. 26.1.2.13d).

Our second objective was to determine as far as possible the course of the polypeptide chain and the positions of the disulfide bridges. This proved to be impossible. In comparison with the maps of myoglobin (Kendrew et al., 1958) and haemoglobin (Perutz et al., 1960) at this resolution, it was immediately apparent that this map of lysozyme had a much smaller proportion of clear-cut rod-like features representing α-helices. This was not a surprise, since optical rotatory dispersion measurements (Yang & Doty, 1957) suggested that only 30–40% of the polypeptide chain in lysozyme is in the form of α-helix, as compared with 77% in myoglobin. In addition, Hamaguchi & Imahori (1964) had distinguished the presence of a region of β-sheet in lysozyme before completion of the X-ray analysis. The task of tracing the polypeptide chain, which was difficult with myoglobin, was impossible with lysozyme, since the connectivity of the non-helical regions was often not discernible. The existence of four disulfide bridges, which were expected to have about the same electron density as helices at this resolution, complicated the problem further.

Accordingly, we concluded that defining the shape of the molecule and its tertiary structure would have to await further studies at higher resolution. Meanwhile, Corey and his colleagues (Stanford et al., 1962) and Dickerson et al. (1962) published interim accounts of their work at the same time as our work was published (Blake et al., 1962).

At this stage, in the autumn of 1962, RJP left for three months for the MRC Laboratory in Cambridge and then joined Howard Dintzis at Johns Hopkins. At about the same time, DFK joined the team to continue the analysis to high resolution, and LNJ joined DCP as a graduate student and began work related to the activity of the enzyme.