Figs. 1 and 2 show views of the ac and bc planes of both the. axes are shown, and inspection of the figure suggests that the

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1 Proc. Nat. Acad. Sci. USA Vol. 71, No. 5, pp , May 1974 The Molecular Structure of Yeast Phenylalanine Transfer RNA in Monoclinic Crystals (molecular replacement method/x-ray diffraction/orthorhombic trna crystal) G. J. QUIGLEY, F. L. SUDDATH, A. McPHERSON, J. J. KIM, D. SNEDEN, AND ALEXANDER RICH Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass Contributed by Alexander Rich, March 14, 1974 ABSTRACT The molecular structure of monoclinic crystals of yeast phenylalanine trna is analyzed by comparing it to the orthorhombic crystal of the same material whose structure has been determined. Changing the packing of the molecule from the head-to-head, tail-totail arrangement in the orthorhombic lattice to a head-totail packing makes it possible to generate a proposed structure for the monoclinic unit cell. The structure factors for the proposed arrangement have been calculated and compared with those experimentally observed from monoclinic crystals. The residuals from this comparison are low enough to conclude that at 4-A resolution, the three-dimensional structure of the molecule in the monoclinic crystal is essentially the same as that in the orthorhombic crystal. In addition, a correlation coefficient calculated from intensities based on a skeletal model of the molecule also confirmed the structure in the monoclinic cell. Electron density difference maps, as well as the presence of close contacts in the anticodon loop region of the monoclinic crystal, suggest that the anticodon loop may have a slightly different conformation than that observed in the orthorhombic crystals. The three-dimensional structure of an orthorhombic crystal of yeast phenylalanine transfer RNA has been determined by x-ray diffraction analysis to a resolution of 3.0 A (1). In that crystal the molecule has an extended L-shaped form in which the anticodon is at one end of the L, the C-C-A acceptor stem is at the other end, and the corner of the L is formed by the folding together of the dihydrouracil and T-4-C loops of the familiar cloverleaf diagram. One of the peculiar features of the crystallography of transfer RNA is the fact that the system is intensely polymorphic; these molecules crystallize in many different forms. There are, for example, a dozen different unit cells in which yeast phenylalanine trna is known to crystallize and these differ not only in the mode of molecular packing but also in the resolution of the x-ray diffraction pattern (2-5). This immediately makes one wonder whether the three-dimensional conformation of the molecule is the same in these various polymorphic forms. Transfer RNA is a polyelectrolyte in which electrostatic repulsions play an important part in determining conformational stability; thus it could be argued that these different modes of packing represent different ways of folding the molecule. In the present paper we carry out an analysis of the x-ray diffraction pattern obtained from the monoclinic crystal form (3-5) and compare it with the orthorhombic crystals. We demonstrate that the molecule has virtually the same conformation in the monoclinic unit cell as it has in the orthorhombic unit cell. The only exception to this may be a small difference near the anticodon end of the molecule. The monoclinic crystal form was chosen for analysis for two reasons. As shown in Table 1, the monoclinic and orthorhombic unit cells are closely related: the a and b axes are identical and the a angle of 900 is preserved, thus the only change is that found in the c axis. In addition, the diffraction pattern produced by the monoclinic crystal form has a high enough resolution to make a comparison informative. Figs. 1 and 2 show views of the ac and bc planes of both the monoclinic and orthorhombic unit cells where an irregular asymmetric figure represents the molecules. The symmetry axes are shown, and inspection of the figure suggests that the monoclinic cell can be generated from the orthorhombic cell by simply changing the molecular packing from the head-to head, tail-to-tail arrangement seen in the orthorhombic lattice to a head-to-tail arrangement seen in the monoclinic. This can be done by taking the shaded pair of molecules on the left of the orthorhombic lattice and inverting them so that the direction of orientation is parallel to that of the molecules on the right of the figure. The molecules can then slide together until the repeat distance corresponds to the 63-A c axis seen in the monoclinic cell. This transformation has the merit of preserving the packing of the molecules along the a axis as well as the 2-fold screw interactions which relate molecules in alternate layers along the b axis of the orthorhombic unit cell. This suggests that the change from orthorhombic to monoclinic packing may be due to a simple rearrangement of the molecules. Here we evaluate this proposed structure for the monoclinic unit cell MATERIALS AND METHODS The monoclinic crystals of yeast phenylalanine trna were prepeared as described (3). They were generally small and imperfectly formed, and many of them were twins. The monoclinic crystals are often distinguished from the orthorhombic by the characteristic striations on the surface of the crystal (3-5). The best specimens were selected, and precession film data were obtained from these at 80C. The projections HKO, HOL, and OKL, as well as HKH and HKK, were collected. Films were scanned on an Optronics film scanner and scaled together to produce a total of 715 unique reflections constituting approximately 37% of the total reflections in the 4.0-A sphere. The orthorhombic crystal data and aspects of the 3-A orthorhombic electron density map have been described (1). The coordinates for the phosphate and ribose groups based on the 3-A orthorhombic map were measured from a model built

2 Proc. Nat. Acad. Sci. USA 71 (1974) P222, P2, I I Molecular Structure of Yeast Phenylalanine trna 2147 o. F ~~~~ ~~161 A ~ *1 LI 3</ 4 FIG. 1. A diagrammatic representation of the packing of trna molecules in the orthorhombic unit cell. Two projections are shown. The upper projection perpendicular to the b axis shows only one layer of molecules; the second layer behind is not shown. Two different sides of the molecules are shown in the lower projection, as indicated by the cross hatching. in an optical comparator. Calculations were carried out on an IBM 370/165. Many of the calculations used the fast Fourier programs of Hubbard, Quicksall, and Jacobson (6). In order to evaluate the proposed monoclinic packing, two procedures were used. A monoclinic map was generated from the orthorhombic electron density map by changing the mode of packing as described in Fig. 1, and this yielded calculated structure factors for the monoclinic structure. These were compared with the observed structure factors. Secondly, coordinates of centers of the phosphate and ribose groups obtained from the orthorhombic cell were used to generate similar coordinates for the monoclinic cell. These were examined for unacceptable contacts and were also used to calculate intensities from which correlation coefficients were computed. In generating the electron density map for the monoclinic cell, we started with a complete orthorhombic electron density map that had been generated from the observed structure factors and most probable phases. A fine grid was used to allow linear interpolation between grid points. The phasing procedure used data described previously (1) but with some additional phase refinement which will be reported elsewhere. The resultant niap is very similar to that described in the paper by Suddath et al. (1) but is computed from unweighted structure factors (F) which are needed to generate unweighted Fs for the monoclinic structure. The packing in the directions along the a and b axes and about the 21 axes parallel to a are assumed to be unchanged in going from the orthorhombic to the monoclinic form. This is shown diagrammatically in Figs. 1 and 2. The electron density corresponding to two neighboring molecules related by the 21 axes along a was left unchanged while the electron densities of other molecules were set equal to the average negative electron density in the orthorhombic cell, roughly the solvent electron density. The electron density corresponding to the volume of the two molecules was then placed in the monoclinic cell so that the 21 axes relating the two molecules were superimposed on the 21 axes along a at z = 0.5. Since the molecules extend beyond the cell limits in z, the molecular envelopes from adjacent unit cells overlap. In these overlap regions the electron density assigned was the be-o C FIG. 2. A diagrammatic representation of the packing trna molecules in the monoclinic unit cell. Only one layer of molecules is shown in the upper projection perpendicular to the b axis. higher electron density from the two molecular volumes. At only a very few points were the overlapping electron densities both positive, indicating no substantial overlap of the molecules. By fast Fourier procedures the monoclinic electron density map was transformed to produce a complete set of monoclinic structure factors to 4.0-A resolution. The various film data were all fit to these calculated structure factors by applying a scale factor of the form A exp(b (sin O/X)2) where 0 is the Bragg angle, X is the wave length, and A and B are constants. The film data were then combined to produce one set of 715 unique observed reflections. From the calculated and observed structure factors, (F0 and Fo), the residuals, R = zjifci - IFoII/lFoI, were calculated as well as projection difference maps having amplitude (1Fol - 1Fc1) and the phase of F,. In order to examine further whether any unacceptable contacts were present, the fractional coordinates of the phosphate and ribose groups of a molecule in the orthorhombic cell were converted into monoclinic fractional coordinates. Using the program ORTEP (7) all intermolecular and intramolecular contacts less than 8 A were computed for the molecules in both the orthorhombic and monoclinic crystal forms. Recently Levitt (8) has suggested the use of the correlation coefficients (CC) to determine agreement between intensities calculated from a crude molecular model (IC) and those observed (Jo), where CC = 100 X Z(IoIC - (Io)(Ic))/[Z(Io2 - ()2)Z(IC2- (IC)2) ]/2. From the coordinates of the phosphate and ribose groups of both the orthorhombic and monoclinic forms of trna, intensities were calculated. Correlation coefficients were then calculated between these intensities and the observed intensities. RESULTS AND DISCUSSION We are carrying out a molecular replacement in which a known structure is being used to interpret an unknown but closely related structure. The close similarity of many of the unit cell parameters of the orthorhombic and monoclinic unit cells (Table 1) suggests a direct method of replacement that preserves two of the three major molecular interactions that build up the known orthorhombic unit cell. These interactions are: (i) the stacking along the a axis in which C-C-A helical stem of one molecule interacts closely with the T-t-C helical stem

3 2148 Biochemistry: Quigley et al. Proc. Nat. Acad. Sci. USA 71 (1974) FIG. 3. Left: A model of the trna molecule as seen at 4-A resolution (9). The various stems and loops of the L-shaped molecule are labeled. Right: The packing of trna molecules along the vertical a axis. Adjacent molecules pack through an interaction between the C-C-C stem of one molecule and the T-V-C stem of its neighbor. This packing is maintained in both the orthorhombic and monoclinic unit cells. of an adjoining molecule. This is seen clearly in Fig. 3, where a three-dimensional molecular model of the structure determined at 4-A resolution (9) is used. (ii) The interactions along the b axis around the 2-fold screw symmetries, which are shown diagrammatically in Figs. 1 and 2; and finally (iii) the packing along the c axis. i and ii are preserved in the monoclinic unit cell, but iii is altered. The packing along the c axis is shown for the orthorhombic lattice in Figs. 1 and 4, and for the monoclinic in Figs. 2 and 5. The molecular models used in Figs. 4 and 5 illustrate the different interactions along the c axis found in the two unit cells. Since we are preserving the 2, axes and only changing the c axis from 161 A to 63 A, it is clear that there are no degrees of freedom in the new packing proposed for the monoclinic unit cell. We next evaluate the correctness of this proposal. The molecular replacement was evaluated in several ways. The residual values (R) from the electron density replacement method are given in Table 2. R values for a structure are influenced by whether or not it has a center of symmetry. For a centrosymmetric distribution, a random collection of atomic positions would produce an R value of 0.83 (10), whereas the proposed monoclinic packing produces an R value of 0.53 in the centric OKL projection. Similarly, for diffraction data that are not centric, a random set of atoms TABLE 1. Crystallographic data for two yeast phenylalanine trna crystals Orthorhombic Monoclinic* Space group P21221 P21 a 33 A 33A b 56A 56i& c 161 X 63AX a 900 * a is chosen here for the monoclinic angle to simplify the notation, would produce an R value of 0.59 (10). However, the observed value for the acentric data in the proposed monoclinic unit cell is These R values suggest that there is a reasonable agreement between the proposed monoclinic structure and the observed structure factors. Some estimate of the agreement can be obtained by comparing these results with other macromolecular structures that have been determined by means of the molecular re placement method. When triclinic lysozyme is compared to the tetragonal form, which was solved by the conventional isomorphous replacement method, an R value of 0.44 was obtained from the 6A data (11) as compared to R = 0.36 for the acentric trna data. A similar molecular replacement study has been reported for the isozyme M4 of lactate dehydrogenase when the dogfish and pig crystal forms were compared. From the 8-A data, there is an overall minimum R value of 0.36 (12), compared to the overall figure of 0.42 for the monoclinic trna at 4-A resolution. It should be noted that all of these residuals are substantially higher than those commonly found for small molecules. This is attributable to many factors, including phasing errors in the original orthorhombic map. The average figure of merit of 0.66 for the orthorhombic data (1) suggests that this alone could account for much of the residual. In addition there is no precise way to TABLE 2. Residual values (R) comparing observed and calculated structure factors for monoclinic crystals Data R* No. of reflections Total Centric (OKL) Acentric HOL HKO HKH HKK * R = ZJIFo[ - 1Fjj /2IFOI.

4 Proc. Nat. Acad. Sci. USA 71 (1974) Molecular Structure of Yeast Phenylalanine trna 2149 FIG. 4. The packing of trna molecules in the orthorhombic unit cell as seen in projection down the b axis. It can be seen that the anticodon loops of two molecules are in close contact around a 2-fold symmetry axis. The symmetry axes are shown in Fig. 1. treat the solvent density in regions of overlap. The procedure used, while reasonable, does introduce some discrepancy in the calculated intensities. Finally, while errors in the measured intensities of the orthorhombic data should be quite small, the monoclinic film data is substantially less accurate with residuals between equivalent films based on the intensities in some cases as high as Another method of comparing a proposed structure with the observed data is to calculate a correlation coefficient comparing a set of intensities predicted from a given set of coordinates with the observed intensities. Correlation coefficients for both the orthorhombic and monoclinic crystal forms were calculated using the backbone ribose and phosphate coordinates as a crude model for the total structure. The orthorhombic data give a correlation coefficient of 26% with an expected standard deviation of 2% for 2,818 reflections. The monoclinic data give a correlation coefficient of 33% with an expected standard deviation of 4% for the 715 observed reflections to a resolution of 4 A. For a random set of atomic coordinates the correlation coefficient would be 0%. Alternatively, a correlation coefficient calculated with all of the atoms and compared with a perfect set of data would give a 100% correlation. The figures 26% and 33% are both sufficiently high to indicate good agreement between the crude model using only the backbone groups of yeast phenylalanine trna and the data from both the orthorhombic and monoclinic unit cells. The higher correlation coefficient for the monoclinic crystal probably arises from the higher percentage of low-resolution data used for that form. A number of close intermolecular contacts exist between the ribose and phosphate groups that are common to both the orthorhombic and monoclinic unit cell. Some of these arise from the close packing of the molecules along the a axis, as shown in Fig. 3, where residues along the backbone of the T-Q-C stem of one molecule are in contact with the C-C-A stem of the molecule directly adjacent to it. Similarly, the contacts between molecules along the b axis, which are related by two-fold screw symmetry, are also retained in both unit cells. In addition there is one intermolecular contact found in FIG..5. The packing of trna molecules in the monoclinic unit cell as seen in projection perpendicular to the b axis. The anticodon loop is seen in close contact with the T-t-C and C-C-A stems of adjoining molecules along the c axis. the orthorhombic unit cell which is absent in the monoclinic. In this contact a phosphate group in the anticodon loop of one molecule is within 8 A of the same phosphate in the anticodon loop of a molecule related to it by the 2-fold axis. The trna molecules in the proposed monoclinic packing have nine pairs of intermolecular contacts within 8 A that are not present in the orthorhombic unit cell. All of these involve the anticodon loop, with two contacts to the C-C-A stem and the remainder to the T-t-C stem of adjacent molecules. Of these contacts only three are shorter than 5.5 A, with a minimum of 4.3 A. Difference electron density maps were calculated along the three principal monoclinic projections. They do not show any major regions of difference. However, there is one common feature, which is a region of negative electron density that appears in the region of the anticodon loop of each map. These differences along with the close contacts involving the anticodon loop suggest that this loop may have a slightly different conformation in the orthorhombic and monoclinic crystals. Unfortunately the difference mraps, which are only in projection, are not sufficiently detailed to allow us to evaluate this suggestion. It is quite clear that further work remains to be done with the monoclinic crystal. A complete set of diffraction data can be obtained up to 3-A resolution and, with proper phasing, a full three-dimensional electron density map can be calculated. This would then allow us to determine in detail the nature of any small differences that are found between the molecules in the monoclinic and orthorhombic unit cells. The results described above clearly indicate that the major conformational features of yeast phenylalanine trna in the orthorhombic unit cell are preserved in the monoclinic unit cell. In both unit cells the molecules have the same extensive contacts with neighboring molecules along the a and b axes but completely different packing in the c direction. The packing along c in the orthorhombic form contains a minimum amount of interaction due to the presence of the large aqueous channels running along the a axis of the crystals (1). Elimination of these aqueous channels are responsible for the observed

5 2150 Biochemistry: Quigley et al. shrinkage behavior of these crystals (13). It is interesting to note that one of these orthorhombic shrinkage forms has a c axis of 128 A, approximately twice the monoclinic c axis. In contrast, the monoclinic crystals appear to have many contacts along the c axis direction. Figs. 4 and 5 show models of the trna packing in the orthorhombic and monoclinic unit cells. The conditions for crystallizing the trna in either the monoclinic or orthorhombic forms do not differ from each other very much (3-5). The fact that the monoclinic crystal form is not strongly preferred and, in fact, seems to give less perfect crystals than the orthorhombic form suggests that some of these contacts may be destabilizing. This may be related to the crowded packing near the anticodon loop of the trna as discussed above. The suggestion has been made by Levitt (8) that the conformation of the trna in the monoclinic crystals dilfers from that of the orthorhombic or that the orthorhombic structure is incorrect. These conclusions appear to arise from a misinterpretation of the nature of the trna structure. His interpretations were based on models such as those shown in Figs While these models show the overall shape of the molecule, it is not possible to determine the helical directions from these with any accuracy. Examination of the 3-A electron density map and model (1) suggest that the correlation coefficient maps of the helical segments that are present (8) are generally consistent with the structure of the molecule. The lack of further interpretable results beyond this point is apparently due to the two-fold axis in the search map causing artificial enhancement (8) as well as the irregularities in the helical regions on the trna, and the nonlinearity of adjacent stems (1). Our interest in the structure of the monoclinic form of yeast phenylalanine trna is related to the general question of the stability of the trna three-dimensional structure and its relative invariance when packed in different crystal lattices. This point is important in developing an understanding of the structure of the molecule not only in the crystalline state but in solution as well. The present results demonstrate that the Proc. Nat. Acad. Sci. USA 71 (1974) overall shape of the molecule is maintained in two different modes of molecular packing. It will, of course, be of interest to determine its structure in other unit cells and ultimately to relate this structure to the three-dimensional structure of other species of transfer RNA. We acknowledge the assistance of Sue Bock in growing crystals and we are indebted to Drs. S. H. Kim, N. C. Seeman, and J. Weinzierl for helpful discussions. We thank Drs. W. Lipscomb and K. Ericks for allowing us to use their optical scanners for measuring the diffraction intensities. G.J.Q. is a fellow of the Medical Foundation while D.S. is supported by an NIH training grant. This research has been supported by grants from the NIH, NSF, NASA, and The American Cancer Society. 1. Suddath, F. L., Quigley, G. J., McPherson, A., Sneden, D., Kim, J. J., Kim, S. H. & Rich, A. (1974) Nature 248, Cramer, F., von der Haar, F., Holmes, K. C., Saenger, W., Schlimme, E. & Schulz, G. E. (1973) J. Mol. Biol. 51, Kim; S. H., Quigley, G., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) J. Mol. Biol. 75, Ichikawa, T. & Sundaralingam, M. (1972) Nature New Biol. 236, Ladner, J. E., Finch, J. T., Klug, A. & Clark, B. F. C. (1972) J. Mol. Biol. 72, Hubbard, C. R., Quicksall, C. 0. & Jacobson, R. A. (1971) Document (Ames Laboratory, USAEC, Iowa State University Ames, Iowa). 7. Johnson, C. K. (1965) Document ORNL-3794, (Oak Ridge National Laboratory, Oak Ridge, Tenn.), revised. 8. Levitt, M. (1973) J. Mol. Biol. 80, Kim, S. H., Quigley, G. J., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) Science 179, Wilson, A. J. C. (1950) Acta Crystallogr. 3, Joynson, M. A., North, A. C. T., Sarma, V. R., Dickerson, R. E. & Steinrauf, L. K. (1970) J. Mol. Biol. 50, Hackert, M. L., Ford, G. C. & Rossmann, M. G. (1973) J. Mol. Biol. 78, Kim, S. H., Quigley, G., Suddath, F. L., McPherson, A., Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) J. Mol. Biol. 75,