Section 26.1.3.10. The electron-density map at 2 Å resolution

GAM joined the team in early 1964 with the specific task of writing a program for calculating the 2 Å electron-density map. Fortunately, at this stage, the University of London Computing Centre was in the course of acquiring a Ferranti ATLAS computer, which was then one of the most powerful computers available for this kind of work, and GAM eagerly set about taking advantage of its power. Whereas we ourselves had operated the previous MERCURY computer at night, there were machine operators for the ATLAS with whom we left our input tapes; these were very large reels of paper tape comprising all of the amplitude and phase data for the 2 Å set. Following two or three unsuccessful attempts to load the whole of the data without tearing the tapes, GAM modified his program so that the data could be loaded in several sections, which was achieved satisfactorily.

The electron density was calculated at 1/120ths of the cell edge along a and b, and 1/60ths along c. The output from the computer was on punched paper tape, arranged with appropriate insertions of carriage returns and line feeds so that the teleprinter output was in a form suitable for immediate contouring to a scale of 0.75 inches equal to 1 Å. Each x, y section of the map was printed out in five strips, which had to be glued together to cover the whole area. The contours were drawn initially in pencil on these paper sheets and were then copied in drawing ink to thin Mylar sheets, which were supported for interpretation on Perspex sheets that were stacked and bolted together, with spacers of appropriate dimensions to maintain the scale in the c direction, in groups of five for ease of handling. The whole map was drawn on 60 sections perpendicular to the z axis, and the bolts holding the blocks of five sheets together were designed to fit into one another to keep successive blocks in register. The maps were viewed on large light boxes, specially constructed for the purpose, though they were not transparent enough for more than fifteen sheets, three blocks of five, to be studied in detail at one time. The contours were drawn on Mylar sheets to avoid waste of the more expensive Perspex that would have arisen from errors. A grid was drawn on a Mylar sheet, which could be superimposed on the contour stacks and used to read the atomic coordinates directly in ångstroms.

The electron density had been calculated using a scale factor such that it was convenient to draw contours at intervals of 0.25 e Å⁻³; as no F(000) term had been included in the calculation, electron-density values were relative to the mean value for the unit cell. Contours were drawn only for electron densities above the mean, the two lowest levels being drawn in orange ink, and the higher ones in black ink. This proved to be a satisfactory form of representation, as it very clearly revealed regions of continuous high electron density while also showing the positions of significant features of lower density. All the members of the lysozyme group participated in drawing the maps.

To illustrate the result that was obtained, sections z = 35/60 to 44/60 of the electron-density map are shown in Fig. 26.1.3.9.

Figure 26.1.3.9| top | pdf | Photograph of sections z = 35/60 to 44/60 of the three-dimensional electron-density map of hen egg-white lysozyme at 2 Å resolution. AA′ shows the axis of a length of α-helix lying in the plane of the sections. B indicates an α-helix more nearly normal to the sections. C indicates the disulfide bridge between residues 30 and 115, the sulfur atoms of which lie one above the other. The side chain of a phenylalanine residue is located four residues along the helix from the disulfide, towards the lower sections. Reproduced with permission from Nature (Blake et al., 1965). Copyright (1965) Macmillan Magazines Limited.