Tuna cytochrome c at 2.0 A resolution. III. Coordinate optimization and comparison of structures.

Optimum coordinate sets have been obtained for ferrocytochrome c and the two symmetry-independent molecules of ferricytochrome c from tuna at 2.0 A resolution by making the best fit of models with standard bond lengths and angles to the experimental electron density maps (1977) J. Biol. Chem. 252, 759-785, as a preliminary to full refinement with 1.5 A data. Both the Diamond model-building programs and locally developed minicomputer routines were tried, with the latter preferred for economy and ease of operation, although both gave satisfactory results. Atomic coordinates are available on microfiche or from the Brookhaven Protein Data Bank. Using the two ferricytochrome molecules as a control, no differences between oxidized and reduced cytochrome molecules can be seen that are outside the probable limits of accuracy of the 2.0 A analysis. Rotation and subtractive difference map comparisons also show no conformation changes. If believable differences do appear in the course of the 1.5 A refinement now underway, these should be no more than minor breathing of main chain or adjustment of side chains.

The crystal structure of oxidized cytochrome c from tuna hearts has been solved by x-ray diffraction to a resolution of 2.0 13, using four isomorphous heavy atom derivatives. The crystals, space group P4:,, have 2 independent cytochrome molecules in the asymmetric repeating unit. No significant difference is seen between these 2 molecules, aside from conformations of a few surface side chains. The molecular folding observed is essentially that reported for tuna ferrocytochrome c. In particular, the ring of phenylalanine 82 lies against the heme group and closes the heme crevice, and is not swung out into the surroundings as had been believed from the 2. 8     heavy atoms is shown in a radial plot in Fig. 11. This quantity is a useful measure of the quality of the phasing contributed by each derivative.
Phasing contributions from platinum cyanide and gold cyanide remained strong to the 2 A limit, but platinum chloride and iridium nitrite shared a high lack of closure error as well as common binding sites at methionine 65. As is discussed later in connection with the mode of binding of heavy atoms, these large errors probably arise from lack of true isomorphism between these two derivatives and the native protein crystals.
The mean residual error or MRE per derivative (17) was close to its expected value of 0.50, indicating that the root mean square error for each derivative in the denominator of the exponential phase probability expression had been properly chosen. Hence, the mean figure of merit in this specific structure analysis is a meaningful assessment of the quality of phasing, which is not always the case when figures of merit are quoted. On this scale the contour interval in the map is 0.18 e/A3, the root mean square error over the entire map is 0.13 e/AZ, and the iron peak height is 5.1 e/A3. A check of this scaling is provided by the observation that the main polypeptide chain density is five to seven contours or about 1.1 e/A3 above average density, a value typical of other protein maps reported at 2 A resolution. The occupancy factors in Table IV and the F data in Table VI can be placed on an absolute scale of electrons per molecule by multiplying by 0.09. The two space group-independent molecules were treated separately throughout the analysis. They are designated as the "inner" molecule, with iron at z = 0.013 in Fig. 9, and the "outer" molecule, with iron at z = 0.004. For model building purposes (Kendrew wire models, 2 cm/A), the electron density region containing each molecule was calculated and displayed in its own two-mirror Richards box of the design shown in Ref. 19. The use of two half-silvered mirrors permitted two maps of the same molecule, sectioned at right angles to one another, to be studied simultaneously as the model was being built. This frequently was useful when a particular feature was oriented unfavorably for viewing in one of the maps. In the cross-sections, the sulfur ligands are prominent because of their density and the heme planes show no obvious sign of nonplanarity.

Overall
The main chain folding in tuna ferricytochrome c is illustrated by front views of the 2 molecules in Fig. 16 and by top views in Fig. 17     at pyrrole rings and minima between rings, is even more pronounced here than in Fig. 14. in the center of Fig. 18. The outer and inner molecules involved in this contact (iron atoms at 0.504 and 0.013 in Fig. 18) are related approximately by a noncrystallographic a-fold screw axis along a cell diagonal in the z = 0.250 plane, with a 10 A screw displacement. Platinum cyanide in site 1 is close to glutamine 16 on the outer molecule and to lysines 7 and 8 and valine 11 on the inner molecule. Site 3 is close to lysines 7 and 8, valine 11, and glutamine 12 on the outer molecule, and to serine 100 and alanine 101 on the inner. Intermolecular packing at this contact interleaves the side chains of the beginning of the NH&rminal helix of the inner molecule, with the end of the corresponding helix in the outer molecule. The packing is nonpolar, with no specific charge interactions. A much looser contact between inner and outer molecules occurs halfway down the z axis, at z = 0.750, again seen in the center of Fig. 18. This also is a nonpolar interaction, with the side chain of asparagine 22 on the inner molecule inserted between histidine 26 and the residue 23-24 main chain of the outer. This contact surface is small and no heavy atoms are trapped between molecules at this point.
If the two contacts just described are labeled A and B, respectively, then tuna ferricytochrome has A-A and B-B con- or "polarity" (in the logical sense) to the mating surfaces.
The outer and inner molecules in Fig. 18   platinum cyanide may be especially valuable precisely because they do not bind strongly to the protein and hence do not introduce distortions and impaired isomorphism. Derivatives with few sites may be necessary in the early stages of an analysis in order to interpret the multisite derivatives, but the latter may be of more value in phasing at high resolution.

Orientation of Aromatic
Rings -Cytochrome c is distinguished by a remarkably conservative set of aromatic residues, which have retained their aromatic character throughout evolutionary history even though the specific amino acids have changed. All positions at which aromatic rings are found in any of the 60 eukaryotic cytochrome sequences from 67 different species are listed in Table VIII, which also shows how many times each position is encountered as phenylalanine, tyrosine, tryptophan, or a nonaromatic group. The 10 aromatic groups in tuna cytochrome are designated in bold face type.
These same key positions tend to remain invariant in the bacterial cytochromes that are known to be structurally homologous with cytochrome c: respiratory cytochrome cssO from Paracoccus denitrificans (8) and photosynthetic c2 from Rhodospirillum rubrum (5) and other purple non-sulfur bacteria. The highly conserved aromatic residues occur in clusters in the molecule, visible in Figs. 16  The pronounced evolutionary conservatism of aromaticity around the heme group suggests that these rings have an important role. Attempts have been made to involve them in electron transfer mechanisms (20,23), although these mechanisms now seem unlikely. No matter what their role, the orientation of these rings and the degree of confidence that can be placed in the published orientations, are matters of interest when the final structures of ferri-and ferrocytochrome c are studied. Numerical comparisons will be deferred until the third paper in this series,2 after the structure of the reduced form of the molecule has been presented.' The immediately comprehensible measure of confidence is the appearance of the electron density map itself. For this reason, miniature inplane sections and cross-sections are shown in Fig. 19 for all histidines and prolines, and in Figs. 20 and 21 for all phenylalanines, tyrosines, and tryptophans.
One generalization is quickly apparent. At 2 A resolution, a five-membered ring appears as a flat disc without a central depression. Histidine 18 in both inner and outer molecules (labeled H18-I and H18-0) are good examples. The angular shape of the disc is correct and the disc cross-section is satisfyingly flat, but there is no central hole or dimple. Prolines 71 and 76 in the outer molecule (P71-0 and P76-0) may be considered especially favorable exceptions to this principle, but the general rule still holds.
In contrast, a six-membered ring is large enough to show a central depression at 2 A resolution, as Figs. 20 and 21 indicate. Phenylalanine 10 in the inner molecule (FlO-I) and tyrosine 46 in both molecules are particularly clear examples and the tyrosines also illustrate the clarity with which an -OH group stands out. Tryptophans 33 and 59 are consistent with the above principles: their six-membered rings are dimpled in the center but their five-membered rings are not.
The ring orientations in the stereo drawings of Figs. 16 and 17 and the plane sections of Figs. 19 to 21 are the results of independent minimap fitting to the inner and outer molecules: hand adjustment of chain positions to map density, alternated with least squares optimization of bond lengths and angles. The ring cross-sections indicate the degree of confidence that can be placed in ring orientation comparisons. For phenylalanine and tyrosine, the uncertainties in rotation about the CD-C, axis should be no more than 5-10". A comparison of the stereo drawings in Figs. 16 and 17 shows that the two symmetry-independent molecules have virtually identical conformations. Aromatic ring orientations are the same within the limits just mentioned. Although some external side chains have slightly different conformations in the inner and outer molecules,2 there is no indication that main chain conformations are influenced by the different local environments that the 2 molecules have in the crystal. The degree of similarity or difference observed in the main chain and internal side chain   Position  1CP  33  35  36  46O  4W  5P  61  54  65  61°  14  320  97'   Tyrosine  1  2  33  60   1   23  59  58  59  Phenylalanine  60  1  2  53  27  1  8  1  2  60  1  Tryptophan  4  60  Other  54  58  5  59  59  portions of these 2 oxidized molecules furnishes a calibration standard or control, against which any presumed folding changes with oxidation state must be compared. Position of Phenylalanine 82 -Phenylalanine 82 is an especially interesting ring in tuna ferricytochrome c because of previously published reports indicating that this residue was positioned differently in the oxidized and reduced molecules (1, 3). These reports were wrong, as can be seen from the composite electron density drawings of Figs. 22 to 24. The sections through the electron density map should be viewed as if they were stacked, jigsawed pieces of plywood, each piece eclipsing from view those below it. Each such piece is outlined by a thick line in Figs. 22 to 24 and higher density contours within a section are represented by thinner lines. All line thicknesses are decreased in sections farther removed from the viewer, as a means of introducing perspective. The view in each figure is from the top, close to that of Fig. 1'7 Plane and cross-sections through aromatic rings in the inner (I) and outer (0) molecules. F = phenylalanine; Y = tyrosine. Chain link lines in plane sections indicate the cutting plane for cross-sections. Note in this figure and the following one that six-membered rings generally are well resolved enough to show a central minimum at 2 A resolution, while five-membered rings are not (Fig. 19).
same features can be seen, At present there is strong crystallodrawing of the equivalent region of the two-derivative, 2.8 A graphic evidence to indicate that the phenylalanine 82 portion resolution horse ferricytochrome map. The resolution of deof the cytochrome c molecule does not undergo any kind of tails is markedly poorer in this map. This is attributable to conformation change when the iron atom is reduced or oxithree causes: the lower nominal resolution of the map, the dized.
smaller number of derivatives used in phase analysis, and the Where did the horse ferricytochrome analysis go astray? poorer intrinsic quality of the horse crystals. Two alternatives The answer can be seen in Fig. 25