Proc. Nat. Acad. Sci. USAVol. 71, No. 5, pp. 2146-2150, May 1974

The Molecular Structure of Yeast Phenylalanine Transfer RNA inMonoclinic 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. 02139

Contributed by Alexander Rich, March 14, 1974

ABSTRACT The molecular structure of monocliniccrystals of yeast phenylalanine tRNA is analyzed by com-paring it to the orthorhombic crystal of the same materialwhose structure has been determined. Changing thepacking of the molecule from the head-to-head, tail-to-tail arrangement in the orthorhombic lattice to a head-to-tail packing makes it possible to generate a proposedstructure for the monoclinic unit cell. The structurefactors for the proposed arrangement have been calculatedand compared with those experimentally observed frommonoclinic crystals. The residuals from this comparisonare low enough to conclude that at 4-A resolution, thethree-dimensional structure of the molecule in the mono-clinic crystal is essentially the same as that in the ortho-rhombic crystal. In addition, a correlation coefficientcalculated from intensities based on a skeletal model ofthe molecule also confirmed the structure in the mono-clinic cell. Electron density difference maps, as well asthe presence of close contacts in the anticodon loop regionof the monoclinic crystal, suggest that the anticodon loopmay have a slightly different conformation than that ob-served in the orthorhombic crystals.

The three-dimensional structure of an orthorhombic crystal ofyeast phenylalanine transfer RNA has been determined byx-ray diffraction analysis to a resolution of 3.0 A (1). In thatcrystal the molecule has an extended L-shaped form in whichthe anticodon is at one end of the L, the C-C-A acceptor stemis at the other end, and the corner of the L is formed by thefolding together of the dihydrouracil and T-4-C loops of thefamiliar cloverleaf diagram. One of the peculiar features of thecrystallography of transfer RNA is the fact that the system isintensely polymorphic; these molecules crystallize in manydifferent forms. There are, for example, a dozen different unitcells in which yeast phenylalanine tRNA is known to crystal-lize and these differ not only in the mode of molecular packingbut also in the resolution of the x-ray diffraction pattern (2-5).This immediately makes one wonder whether the three-dimen-sional conformation of the molecule is the same in these vari-ous polymorphic forms. Transfer RNA is a polyelectrolyte inwhich electrostatic repulsions play an important part in deter-mining conformational stability; thus it could be argued thatthese different modes of packing represent different ways offolding the molecule. In the present paper we carry out ananalysis of the x-ray diffraction pattern obtained from themonoclinic crystal form (3-5) and compare it with the ortho-rhombic crystals. We demonstrate that the molecule has vir-tually the same conformation in the monoclinic unit cell as ithas in the orthorhombic unit cell. The only exception to thismay be a small difference near the anticodon end of the mole-cule.

The monoclinic crystal form was chosen for analysis for tworeasons. As shown in Table 1, the monoclinic and orthorhombicunit cells are closely related: the a and b axes are identicaland the a angle of 900 is preserved, thus the only change isthat found in the c axis. In addition, the diffraction patternproduced by the monoclinic crystal form has a high enoughresolution to make a comparison informative.

Figs. 1 and 2 show views of the ac and bc planes of both themonoclinic and orthorhombic unit cells where an irregularasymmetric figure represents the molecules. The symmetryaxes are shown, and inspection of the figure suggests that themonoclinic cell can be generated from the orthorhombic cellby simply changing the molecular packing from the head-tohead, tail-to-tail arrangement seen in the orthorhombic latticeto a head-to-tail arrangement seen in the monoclinic. Thiscan be done by taking the shaded pair of molecules on the leftof the orthorhombic lattice and inverting them so that thedirection of orientation is parallel to that of the molecules onthe right of the figure. The molecules can then slide togetheruntil the repeat distance corresponds to the 63-A c axis seen inthe monoclinic cell. This transformation has the merit ofpreserving the packing of the molecules along the a axis as wellas the 2-fold screw interactions which relate molecules inalternate layers along the b axis of the orthorhombic unit cell.This suggests that the change from orthorhombic to mono-clinic packing may be due to a simple rearrangement of themolecules. Here we evaluate this proposed structure for themonoclinic unit cell.

MATERIALS AND METHODS

The monoclinic crystals of yeast phenylalanine tRNA wereprepeared as described (3). They were generally small andimperfectly formed, and many of them were twins. The mono-clinic crystals are often distinguished from the orthorhombicby the characteristic striations on the surface of the crystal(3-5). The best specimens were selected, and precession filmdata 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 scaledtogether to produce a total of 715 unique reflections constitut-ing approximately 37% of the total reflections in the 4.0-Asphere.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 onthe 3-A orthorhombic map were measured from a model built

2146

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Molecular Structure of Yeast Phenylalanine tRNA 2147

P222, P2,

o.

F ~~~~ ~~~161A *1

LI 3</ 4

FIG. 1. A diagrammatic representation of the packing oftRNA molecules in the orthorhombic unit cell. Two projectionsare shown. The upper projection perpendicular to the b axisshows only one layer of molecules; the second layer behind isnot shown. Two different sides of the molecules are shown in thelower 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 Fourierprograms of Hubbard, Quicksall, and Jacobson (6).

In order to evaluate the proposed monoclinic packing, twoprocedures were used. A monoclinic map was generatedfrom the orthorhombic electron density map by changing themode of packing as described in Fig. 1, and this yielded cal-culated structure factors for the monoclinic structure. Thesewere compared with the observed structure factors. Secondly,coordinates of centers of the phosphate and ribose groupsobtained from the orthorhombic cell were used to generatesimilar coordinates for the monoclinic cell. These were exam-

ined for unacceptable contacts and were also used to calculateintensities from which correlation coefficients were computed.

In generating the electron density map for the monocliniccell, we started with a complete orthorhombic electron densitymap that had been generated from the observed structurefactors and most probable phases. A fine grid was used toallow linear interpolation between grid points. The phasingprocedure 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 thepaper by Suddath et al. (1) but is computed from unweightedstructure factors (F) which are needed to generate unweightedFs for the monoclinic structure. The packing in the directionsalong the a and b axes and about the 21 axes parallel to a are

assumed to be unchanged in going from the orthorhombic tothe monoclinic form. This is shown diagrammatically in Figs.1 and 2. The electron density corresponding to two neighbor-ing molecules related by the 21 axes along a was left unchangedwhile the electron densities of other molecules were set equal tothe average negative electron density in the orthorhombic cell,roughly the solvent electron density. The electron densitycorresponding to the volume of the two molecules was thenplaced in the monoclinic cell so that the 21 axes relating thetwo molecules were superimposed on the 21 axes along a atz = 0.5. Since the molecules extend beyond the cell limits in z,

the molecular envelopes from adjacent unit cells overlap. Inthese overlap regions the electron density assigned was the

be-oC

FIG. 2. A diagrammatic representation of the packing tRNAmolecules in the monoclinic unit cell. Only one layer of moleculesis shown in the upper projection perpendicular to the b axis.

higher electron density from the two molecular volumes. Atonly a very few points were the overlapping electron densitiesboth positive, indicating no substantial overlap of the mole-cules. By fast Fourier procedures the monoclinic electrondensity map was transformed to produce a complete set ofmonoclinic structure factors to 4.0-A resolution.The various film data were all fit to these calculated struc-

ture factors by applying a scale factor of the form A exp(B(sinO/X)2) where 0 is the Bragg angle, X is the wave length, and Aand B are constants. The film data were then combined toproduce one set of 715 unique observed reflections. From thecalculated and observed structure factors, (F0 and Fo), theresiduals, R = zJIFCI - IFoII/lFoI, were calculated as well asprojection difference maps having amplitude (1Fol - 1Fc1)and the phase of F,.

In order to examine further whether any unacceptable con-tacts were present, the fractional coordinates of the phosphateand ribose groups of a molecule in the orthorhombic cell wereconverted into monoclinic fractional coordinates. Using theprogram ORTEP (7) all intermolecular and intramolecularcontacts less than 8 A were computed for the molecules inboth the orthorhombic and monoclinic crystal forms.

Recently Levitt (8) has suggested the use of the correlationcoefficients (CC) to determine agreement between intensitiescalculated from a crude molecular model (IC) and thoseobserved (Jo), where CC = 100 X Z(IoIC- (Io)(Ic))/[Z(Io2 -()2)Z(IC2- (IC)2)]/2. From the coordinates of the phosphateand ribose groups of both the orthorhombic and monoclinicforms of tRNA, intensities were calculated. Correlation co-efficients were then calculated between these intensities andthe observed intensities.

RESULTS AND DISCUSSION

We are carrying out a molecular replacement in which a knownstructure is being used to interpret an unknown but closelyrelated structure. The close similarity of many of the unit cellparameters of the orthorhombic and monoclinic unit cells(Table 1) suggests a direct method of replacement that pre-serves two of the three major molecular interactions that buildup the known orthorhombic unit cell. These interactions are:(i) the stacking along the a axis in which C-C-A helical stemof one molecule interacts closely with the T-t-C helical stem

Proc. Nat. Acad. Sci. USA 71 (1974)

I I

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

2148 Biochemistry: Quigley et al.

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 theC-C-C stem of one molecule and the T-V-C stem of its neighbor. This packing is maintained in both the orthorhombic and monoclinicunit cells.

of an adjoining molecule. This is seen clearly in Fig. 3, where a

three-dimensional molecular model of the structure deter-mined at 4-A resolution (9) is used. (ii) The interactions alongthe b axis around the 2-fold screw symmetries, which are

shown diagrammatically in Figs. 1 and 2; and finally (iii) thepacking along the c axis. i and ii are preserved in the mono-

clinic unit cell, but iii is altered. The packing along the c axisis shown for the orthorhombic lattice in Figs. 1 and 4, and forthe monoclinic in Figs. 2 and 5. The molecular models used inFigs. 4 and 5 illustrate the different interactions along the c

axis found in the two unit cells. Since we are preserving the2, axes and only changing the c axis from 161 A to 63 A, it isclear that there are no degrees of freedom in the new packingproposed for the monoclinic unit cell. We next evaluate thecorrectness of this proposal.The molecular replacement was evaluated in several ways.

The residual values (R) from the electron density replace-ment 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 ofatomic positions would produce an R value of 0.83 (10),whereas the proposed monoclinic packing produces an Rvalue of 0.53 in the centric OKL projection. Similarly, fordiffraction data that are not centric, a random set of atoms

TABLE 1. Crystallographic data for twoyeast phenylalanine tRNA crystals

Orthorhombic Monoclinic*

Space group P21221 P21

a 33 A 33Ab 56A 56i&c 161 X 63AXa 900

* a is chosen here for the monoclinic angle to simplify the nota-tion,

would produce an R value of 0.59 (10). However, the observedvalue for the acentric data in the proposed monoclinic unitcell is 0.36. These R values suggest that there is a reasonableagreement between the proposed monoclinic structure and theobserved structure factors.Some estimate of the agreement can be obtained by com-

paring these results with other macromolecular structuresthat have been determined by means of the molecular re place-ment method. When triclinic lysozyme is compared to thetetragonal form, which was solved by the conventional iso-morphous replacement method, an R value of 0.44 wasobtained from the 6A data (11) as compared to R = 0.36 forthe acentric tRNA data. A similar molecular replacementstudy has been reported for the isozyme M4 of lactate dehy-drogenase when the dogfish and pig crystal forms were com-pared. From the 8-A data, there is an overall minimum Rvalue of 0.36 (12), compared to the overall figure of 0.42 forthe monoclinic tRNA at 4-A resolution. It should be notedthat all of these residuals are substantially higher than thosecommonly found for small molecules. This is attributable tomany factors, including phasing errors in the original ortho-rhombic map. The average figure of merit of 0.66 for theorthorhombic data (1) suggests that this alone could accountfor much of the residual. In addition there is no precise way to

TABLE 2. Residual values (R) comparing observed andcalculated structure factors for monoclinic crystals

Data R* No. of reflections

Total 0.42 715Centric (OKL) 0.53 328Acentric 0.36 387HOL 0.43 135HKO 0.29 104HKH 0.38 146HKK 0.30 93

* R = ZJIFo[ - 1Fjj|/2IFOI.

Proc. Nat. Acad. Sci. USA 71 (1974)

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Molecular Structure of Yeast Phenylalanine tRNA 2149

FIG. 4. The packing of tRNA molecules in the orthorhombicunit cell as seen in projection down the b axis. It can be seen thatthe anticodon loops of two molecules are in close contact arounda 2-fold symmetry axis. The symmetry axes are shown in Fig. 1.

treat the solvent density in regions of overlap. The procedureused, while reasonable, does introduce some discrepancy inthe calculated intensities. Finally, while errors in the mea-

sured intensities of the orthorhombic data should be quitesmall, the monoclinic film data is substantially less accuratewith residuals between equivalent films based on the intensi-ties in some cases as high as 0.15.Another method of comparing a proposed structure with

the observed data is to calculate a correlation coefficient com-

paring a set of intensities predicted from a given set of co-

ordinates with the observed intensities. Correlation coeffi-cients for both the orthorhombic and monoclinic crystal formswere calculated using the backbone ribose and phosphate co-

ordinates as a crude model for the total structure. The ortho-rhombic data give a correlation coefficient of 26% with an ex-

pected standard deviation of 2% for 2,818 reflections. Themonoclinic data give a correlation coefficient of 33% with an

expected standard deviation of 4% for the 715 observed re-

flections to a resolution of 4 A. For a random set of atomiccoordinates the correlation coefficient would be 0%. Alter-natively, a correlation coefficient calculated with all of theatoms and compared with a perfect set of data would give a

100% correlation. The figures 26% and 33% are both suffi-ciently high to indicate good agreement between the crudemodel using only the backbone groups of yeast phenylalaninetRNA and the data from both the orthorhombic and mono-

clinic unit cells. The higher correlation coefficient for themonoclinic crystal probably arises from the higher percentageof 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 theorthorhombic 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 theT-Q-C stem of one molecule are in contact with the C-C-Astem of the molecule directly adjacent to it. Similarly, thecontacts between molecules along the b axis, which are relatedby two-fold screw symmetry, are also retained in both unitcells. In addition there is one intermolecular contact found in

FIG. .5. The packing of tRNA molecules in the monoclinicunit cell as seen in projection perpendicular to the b axis. Theanticodon loop is seen in close contact with the T-t-C and C-C-Astems 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 onemolecule is within 8 A of the same phosphate in the anticodonloop 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 arenot present in the orthorhombic unit cell. All of these involvethe anticodon loop, with two contacts to the C-C-A stem andthe remainder to the T-t-C stem of adjacent molecules. Ofthese contacts only three are shorter than 5.5 A, with a mini-mum of 4.3 A.

Difference electron density maps were calculated along thethree principal monoclinic projections. They do not show anymajor regions of difference. However, there is one commonfeature, which is a region of negative electron density thatappears in the region of the anticodon loop of each map.These differences along with the close contacts involving theanticodon loop suggest that this loop may have a slightly dif-ferent conformation in the orthorhombic and monocliniccrystals. Unfortunately the difference mraps, which are onlyin projection, are not sufficiently detailed to allow us to eval-uate this suggestion.

It is quite clear that further work remains to be done withthe monoclinic crystal. A complete set of diffraction data canbe obtained up to 3-A resolution and, with proper phasing, afull three-dimensional electron density map can be calculated.This would then allow us to determine in detail the nature ofany small differences that are found between the molecules inthe monoclinic and orthorhombic unit cells.The results described above clearly indicate that the major

conformational features of yeast phenylalanine tRNA in theorthorhombic unit cell are preserved in the monoclinic unitcell. In both unit cells the molecules have the same extensivecontacts with neighboring molecules along the a and b axes butcompletely different packing in the c direction. The packingalong c in the orthorhombic form contains a minimum amountof interaction due to the presence of the large aqueous chan-nels running along the a axis of the crystals (1). Eliminationof these aqueous channels are responsible for the observed

Proc. Nat. Acad. Sci. USA 71 (1974)

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

2150 Biochemistry: Quigley et al.

shrinkage behavior of these crystals (13). It is interesting tonote that one of these orthorhombic shrinkage forms has a caxis of 128 A, approximately twice the monoclinic c axis. Incontrast, the monoclinic crystals appear to have many con-tacts along the c axis direction. Figs. 4 and 5 show models ofthe tRNA packing in the orthorhombic and monoclinic unitcells. The conditions for crystallizing the tRNA in either themonoclinic or orthorhombic forms do not differ from eachother very much (3-5). The fact that the monoclinic crystalform is not strongly preferred and, in fact, seems to give lessperfect crystals than the orthorhombic form suggests thatsome of these contacts may be destabilizing. This may berelated to the crowded packing near the anticodon loop of thetRNA as discussed above.The suggestion has been made by Levitt (8) that the con-

formation of the tRNA in the monoclinic crystals dilfers fromthat of the orthorhombic or that the orthorhombic structureis incorrect. These conclusions appear to arise from a mis-interpretation of the nature of the tRNA structure. His inter-pretations were based on models such as those shown in Figs.3-5. While these models show the overall shape of the mole-cule, it is not possible to determine the helical directions fromthese with any accuracy. Examination of the 3-A electrondensity map and model (1) suggest that the correlation co-efficient maps of the helical segments that are present (8) aregenerally consistent with the structure of the molecule. Thelack of further interpretable results beyond this point is ap-parently due to the two-fold axis in the search map causingartificial enhancement (8) as well as the irregularities in thehelical regions on the tRNA, and the nonlinearity of adjacentstems (1).Our interest in the structure of the monoclinic form of

yeast phenylalanine tRNA is related to the general questionof the stability of the tRNA three-dimensional structure andits relative invariance when packed in different crystal lattices.This point is important in developing an understanding of thestructure of the molecule not only in the crystalline state butin solution as well. The present results demonstrate that the

overall shape of the molecule is maintained in two differentmodes of molecular packing. It will, of course, be of interest todetermine its structure in other unit cells and ultimately torelate this structure to the three-dimensional structure of otherspecies of transfer RNA.

We acknowledge the assistance of Sue Bock in growing crys-tals and we are indebted to Drs. S. H. Kim, N. C. Seeman, andJ. Weinzierl for helpful discussions. We thank Drs. W. Lipscomband K. Ericks for allowing us to use their optical scanners formeasuring the diffraction intensities. G.J.Q. is a fellow of theMedical Foundation while D.S. is supported by an NIH traininggrant. 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, 20-24.

2. Cramer, F., von der Haar, F., Holmes, K. C., Saenger, W.,Schlimme, E. & Schulz, G. E. (1973) J. Mol. Biol. 51, 523-530.

3. Kim; S. H., Quigley, G., Suddath, F. L., McPherson, A.,Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) J.Mol. Biol. 75, 421-428.

4. Ichikawa, T. & Sundaralingam, M. (1972) Nature New Biol.236, 174-175.

5. Ladner, J. E., Finch, J. T., Klug, A. & Clark, B. F. C. (1972)J. Mol. Biol. 72, 99-101.

6. Hubbard, C. R., Quicksall, C. 0. & Jacobson, R. A. (1971)Document 15-2625 (Ames Laboratory, USAEC, Iowa StateUniversity Ames, Iowa).

7. Johnson, C. K. (1965) Document ORNL-3794, (Oak RidgeNational Laboratory, Oak Ridge, Tenn.), revised.

8. Levitt, M. (1973) J. Mol. Biol. 80, 255-263.9. Kim, S. H., Quigley, G. J., Suddath, F. L., McPherson, A.,

Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973)Science 179, 285-288.

10. Wilson, A. J. C. (1950) Acta Crystallogr. 3, 397-398.11. Joynson, M. A., North, A. C. T., Sarma, V. R., Dickerson,

R. E. & Steinrauf, L. K. (1970) J. Mol. Biol. 50, 137-142.12. Hackert, M. L., Ford, G. C. & Rossmann, M. G. (1973) J.

Mol. Biol. 78, 665-673.13. Kim, S. H., Quigley, G., Suddath, F. L., McPherson, A.,

Sneden, D., Kim, J. J., Weinzierl, J. & Rich, A. (1973) J.Mol. Biol. 75, 429-432.

Proc. Nat. Acad. Sci. USA 71 (1974)

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0