Electron microscopy of tRNA crystals

J. Mol. Biol. (1984) 172, 347-354

Electron Microscopy of tRNA Crystals 11.I" 4 A, Resolution Diffraction Pattern and Substantial Stability to Radiation Damage

I".lectron microscopy was applied to thin crystals of yeast tRNAPhL The crystals embedded in glucose yiehl Bragg reflections with a spacing smaller than 4 A. The measurement of radiation damage rate demonstrates that they are 4 to 14 times le~s suscel)tible to electron exposures than protein crystals embedded in glucose.

In recent )'ears electron microscopy has become a I)owerfld tool for the study of the tertiary structure of proteins (Amos et al., 1982). However, apart from a preliminary investigation on the fi'ozen lamellar crystal of I)NA (Downing & Glaeser, 1980), no apl)lieati()n of electron microscol)y has as yet been made to l~olymlclcotitle crystals, in contrast to a large number of investigations of l)rotein crystals. We have undertaken electron microscol)ic investigations on the 3"east tl{NA Phe crystals as a representative of various polynucleotide crystals to be subjected to electron microscopy for structural study in the fllture. The monoclinic form of the yeast tRNA T M crystals is most suitable for the investigation of l)roblems such as the distortion of molecules, image contrast and intrinsic regularities of crystals, since its detailed three-dimensional structure h.as been established i)y X-ray crystallographic analysis (Ladner et at., 1975). Our l)reliminary stu(ly has already led to some conclusions about the characteristies of the image from the crystal negatively stained with uranyl acetate, although the staining resulted in lowering the regularity to the 13 A resolution level (Fujiyoshi et al., 1983). x,'lre describe here the successfid application of a glucose embedding technique (Unwin & llen(lerson, 1975) to tRNA crystals that may make it possible to analyse the structure at a resolution better than 4 A, and also the substantial stability of the crystal embedded in glucose to electron exl)osures as coml)arcd with l)rotcin crystals embedded in glucose. (a) Specimen preparation and electron diffraction Crystals of yeast tRNA the sufficiently thin for electron microscopy were grown as described (Fujiyosl~i et at., 1983). For the embednmnt of specimen crystals in glucose, they were soaked into tile crystallizing solution containing 6 to 11% (w/v) glucose at 5~ for 15 hours. A droplet of the crystallizing solution was placed on a microgrid with small boles of several micrometres diameter (Fukami & Adachi, 1965} and then sucked through the grid holes with pieces of tilter l)aper t I'aper I in this series is Fujiyoshi et al. (1983). 347 0022-2836/84/030347-08 $03.00/0

0 1984 Academic Press Inc. (London) Ltd.

Flo. I. (a) Electron micrograph showing yeast t R N A TM crystals embedded in glucose and fixed on a microgrid. Dark and bright areas correspond to pieces of th~ crystals and clefts, respectively. (b) Representative of electron diffraction patterns from yeast tRNA rhc crystals embedded in glucose. The arrow indicates a diffraction spot corresponding to a Bragg spacing of 3 A. Diffuse arcs with the maximum at about 3.4 A are also seen.

LETTERS TO TIIE EDITOR

"34!)

so as to fix tile microcrystals steadily on the grid surface. The grid with crystals was dried in a desiccator at 5~ for more than an hour before observation in an electron microscope. Figure l(a) shows crystals mounted on the grid; they are visible as clark regions. Some larger crystals appear to have heen cracked into more and smaller pieces, possibly because of shrinkage during specimen preparation. The cracking perhaps accounts for the edges of the crystals that are not straight. Electron diffraction patterns were obtained with a JEM-200CX electron microscope equipped with a minimum dose system. It was usually operated at 200 kV, corresponding to a wavelength of 0.025 A. Crystals of an appropriate thickness were searched under a low-dosage condition of about 0.1 e/A 2. For crystals embedded in glucose, electron diffraction patterns were taken by exposure to an electron beam of 7.1 • l0 - s A/cm 2 for 32 to 64seconds. The camera length was selected to be 104 cm or 160 cm. Figure l(b) shows an electron diffraction pattern with a resolution better than 4 A. Some spots were occasionally observed up to 3 A Bragg spacing, as indicated with an arrow in the Figure. The camera constant was calibrated by using a spacing (2.35 A) with the (11 l) diffraction ring from gold crystals, which covered the grids. The dimensions of the unit cell were thus determined to he a = 52 A and b = 32 A. The lengths were found to shrink slightly in both axes as compared with the data for the native crystal from X-ray diffraction; i.e. a = 56 A, b = 33.4 A, c = 63 A, fl = 90"4 ~ and space group P21 (Ladner el al., 1972). The extinction law o f k = 2n was well-preserved, as seen in the diffraction pattern. The image of the negatively stained crystals confirms that tile tRNA molecules are more closely packed along the b-axis and the wide water-rich regions are present between molecules along the same axis. This seems to account for the larger shrinkage in the dimension of the a-axis (4 A) than in that of b-axis (1.4 A). Tile dimensions of tile stained crystals were estimated at a = 54 ( + l ) A, b - - 33.5 (_+0.5) A and c = 63.5 (_+0.5) A. They are close to those of the unit cell dimensions of the native crystal. We therefore believe that the dimension of the c-axis remained similar to that of the native crystal after the treatment with glucose, although it was not measured directly by using a tilting specimen holder. When the intensities on the (hkO) plane were compared between electron diffraction (Fig. l(b)) an(l X-ray diffraction (see Plate II in the paper by Ladner et al., 1972), the relative intensities of the electron diffraction were found to be similar to those obtained by X-ray diffraction beyond 7 A but they seemed to be more different in the region below 7 A. This tendency is consistent with the findings so far obtained for 1)rotein crystals embedded in glucose. However, the distinctive boundary of resolution seems to be somewhat higher than that of protein crystals (10A), perhaps because of the spacings of phosphates in polynucleotide backbones. Together with the preservation of tile crystal lattice, it suggests that the conformation of tRNA molecules was not greatly changed within the crystal embedded in glucose. The diffraction spots in Figure l(b) are not completely sharp, especially at higher resolution. The hlurring is possibly due to uneven shrinkage or lack of flatness in the specimen, which occurred during dehydration. The apparent

350

Y.l.'UJIYOSlll ET AL.

temperature factor of the electron diffraction pattern appears to be greater than that of the X-ray diffraction l)attern, indicating that disorder within the crystal increased during specimen prel)aration. The value of the tcml)erature factor seemed to depend rather sensitively upon conditions for sl)eeimen prei)aration such as glucose concentrations or the temperature during dehydration. The diffraction l)atterns also exhibited some diffuse arcs oriented in the direction of [110]. The spacing of the strongest arc was about 3.4 A. These diffuse reflections were ol)viously derived from the stacked bases in double-helical regions of the tRNA molecules. It was found possible to use sucrose at a similar concentration as an embe(lding medium instead of glucose, although it yielded electron diffraction patterns with a somewhat lower resolution, of about 5 ~1,. We have also tried to embed the crystals in Au-thioglucose. In this case, the suitable concentration for soaking ranged from 5 to 6% (w/v), which was lower than that for glucose. For the moment, the best resolution of the crystals embedded in Au-thioglucose is al)out 8 A. From the electron diffraction pattern of the (hkO) i)lane, the dimensions of the a- and b-axes were estimated at 48 A and ~ respectively, indicating the larger shrinkages in both dimensions. This larger change of unit cell dimensions may have caused the considerable disorder in the crystals. (b) Measurement of radiation damage Biological molecules are highly suscel)tible to radiation damage by electron irradiation. For l)roteins such as imcteriorhodopsin and catalase, the doses of 0.5 e]• ~ or less would be more al)l)ropriate for structural analysis. It is of interest to compare the degree of radiation damage in tRNA crystals with that in I)rotein crystals, since no quantitative measurement of the radiation damage has so far been made for polynuclcotide crystals. The radiation damage of tRNA crystals was measured by monitoring the fading of the electron diffraction patterns fl'om crystals embedded in different media at increasing electron exI)osures. If specimen heating can be neglected, the radiation damage dei)ends on the numi)er of incident electrons per unit area. To estimate the number of incident electrons fed to the crystals, the current density on a focus screen was measured with a /t/t-ammeter attached to the JEM-200CX instrument both before and after taking a series of electron diffraction 1)atterns from the specimen crystals. The ammeter and the image magnification were calil)rated in advance with a Farrady Cage (JEOL, EM-SCF) and a carl)on cross-grating rel)lica, resI)cctivcly. The intensities of diffraction spots in the (hkO) I)lane were digitized with an optical densitometer (Rigaku, 5IJ800I~B). It was not possible to measure tile intensities of Bragg reflections and the unseattercd electron I)eam simultaneously with a single device because the differences between the two intensities were too large. The diffraction intensities were therefore scaled between different fihns by normalizing to the intensity of the Bragg reflection (111) of gold crystals. These relative intensities were I)lotted against the dose of 200 kV electrons to the crystals. Figure 2 tel)resents the decay curves for several randomly chosen reflections from the crystals embedded in

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Fla. 2. l)ecay curves of the intensities of various Bragg reflections. Indices are shown in the all)babette order from a to t as follows: (1 0 0), (2 0 0), (2 1 0), (3 0 0), (4 0 0), (4 1 0), (4 2 0), (G i 0), (1 4 0), (7 0 0), (I 5 0), (7 3 0), (9 0 0), (0 6 0), (10 0 0), (10 2 0), (11 0 0), (2 7 0), (12 0 0), (12 2 0). (a) tRNA crystals embedded in glucose: 200 EV irradiation. (b) t R X A crystals embedded in Au-thioglucose: 200 kV irradiation. (c) tRNA crystals stained with 29o (w/v) uranyl at~etate solution: 200 kV irradiation. Tim number of ineldcnt electrons is expressed in terms of 100 x scale as eoml)ared with those of (a) and (b).

352

Y. FUJIYOSHI E T A L .

glucose and Au-thioglucose as well as those from the crystals negatively stained with 2~ (w/v) uranyl acetate. Most reflections were found to decay exponentially, to a good approximation, regardless of the method used for specimen preservation. In the case of crystals embedded in glucose, the diffuse arcs arising from double-helical regions of the molecules faded at a rate similar to that in Bragg reflections. The critical dose De is a commonly used parameter to specify the radiation damage of different specimens and it is defined as the dose at which a diffraction intensity falls to l/e (37~o) of its initial value. The critical dose for the tRNA cryslals embedded in Au-thioglucose is approximately half that for those embedded in glucose. This instability of the Au-thioglucose-embedded crystal is possibly due to tile disorder increasing within the crystal, as indicated by their low-resolution diffraction pattern. The crystals stained with uranyl acetate yield a critical dose value about 140 times larger than those embedded in glucose, indicating an increased stability similar to that of protein crystals. Table 1 summarizes the critical doses of the tRNA crystals embedded in glucose as evaluated from decay curves of Bragg reflections with spacings smaller than 5.6 A. Tbe critical doses of two protein crystals so far reported are given in the Table for comparison. We defined a gain factor: G = 0.63Dr162

in order to specify the stability of tRNA crystals in comparison with protein crystals. Tile factor of 0.63 was introduced to refer De values of tRNA crystals at 200 kV to those of protein crystals at 100 kV according to the stopping power theory (Bethe, 1933), where the variation of critical dose follows f12 dependence (fl=electron velocity]light velocity). Though the experiments of electron diffraction on L-valine crystals indicated 173 dependence (Howitt et al., 1976) rather than 172 dependence as expected from tile stopping power theory, the value of the factor was not changed greatly by assuming the f13 dependence. As shown in Table l, the critical doses for the catalase crystal and the purple membrane embedded in glucose range from 0.5 to 1-5 c/A 2, the values of which were evaluated from the decay curves already reported (Unwin & Henderson, 1975). The critical dose of thin crotoxin complex crystals embedded in glucose was also measured at 300 kV and found to range between 0.3 and 1.0 e/A 2 with a mean value of 0.5 e/A 2 from the Bragg reflections with spacings smaller than 6 A (Chili et al., 1981). On the other hand, the minimum critical dose of the tRNA crystals embedded in glucose was found to be more than 1.9 e/A 2 (corrected for 100kV), which was evaluated from the curve with the highest decay rate (Fig. 2(a)). The gain of the tRNA crystals to the protein crystals was thus estimated at 4 when compared with the respective highest decay rates. For the Bragg reflections with spacings smaller than 5-6 A, the gain to catalase was found to range from 5 to 14 with a mean value of 9. We therefore conclude that tRNA crystals embedded in glucose were about ten times less susceptible to electron exposures at room temperature than the protein crystals "embedded in glucose. The fading of electron diffraction patterns is attributed to the loss of long-range order within crystals and hence it may be related to the strength of the bonds

LETTERS TO TIlE EDITOR

353

TaBLF 1

Comparison of the stabilities to radiation damage between tRNA and protein crystals embedded in ghtcose Materials Reflecting indexes Spacing (A} Critical dose (e/~?) (Accelerating voltage) Corrected values (Accelerating voltage) O~

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Gc and r were modified by the factor 0"63 (see the formula in the text) to take into account the difference of voltages operated for the tRNA and the protein crystals. (7~: ratio of the critical dose of the tRNA crystal to that of the catalase crystal based on the Bragg reflection (0 38). Gp: ratio of the critical dose of the tRNA crystal to that of the purple membrane based on the Bragg retlection (5 3).

stabilizing the molecular assemblies. I t is known t h a t most t R N A crystals so far obtained have higher water contents than protein crystals. Although tire t R N A crystals used ill this s t u d y contain 63~o water, which is close to tile minimum water content a m o n g the various t R N A crystals, tim value is still higher than those of most I)rotein crystals. I t is believed by X - r a y crystallographers t h a t t R N A crystals, are, in general, more sensitive to X - r a y irradiation, heat and mechanical force than protein crystals. In addition, tim unit cell dimensions of the crystals used here depends sensitively on the t r e a t m e n t for embedding in glucose or Au-thioglucose, indicating relatively weak association between the molecules within the t R N A crystals. These facts suggest t h a t the lower suscelltibility of the t R N A crystals to electron exposures is not due to tile bonds stabilizing molecular assemblies. Meanwhile, it is probable t h a t the stability is related to the aromatic bases within t R N A molecules. The characteristic electron energy spectra or tim mass loss measured on the various biological substances suggested t h a t the valence shell ionization leads to tile break-up of non-conjugated aliphatic molecular components, whereas a r o m a t i c coml)onents require the K-shell ionization to be disrupted (Isaacson, 1973). Ill fact, a significant correlation was found between the characteristic doses of tile radiation d a m a g e and the relative resonance energy per electron from tile m e a s u r e m e n t of tim low-lying (0 to l0 eV) electron energy loss spectra on nucleic acid bases (Isaacson et al., 1973) as well as aromatic amino acids (Lin, 1974). Tire loss of mass due to electron irradiation was also negligible w i t h i n experimental error for tim nucleic acid bases (Isaacson et al., 1973). Furthermore, the degree of radiation d a m a g e is dependent upon tile atomic numbers. The ratio of the elastic cross-section to the inelastic one is 2"3-fold larger in tim phosphorus a t o m than in tire carbon a t o m ( l s a a c s o n , 1977). Tile presence of phosphate groups in t R N A molecules could contribute additionally to the substantial stability observed in this study.

:~54

Y. FUJIYOSlll ET AL.

Institute for Chemical Research Kyoto University, Uji Kyoto-Fu 61 l, Jal)an

Y. FuJiYOSlll

N. UVEDA

Department of Biol)hysics Faculty of Science Kyoto University Kyoto 606, ,lal)an

K. 3IoRIKAWAt It. YAMAOISIII

Received 6 October 1983 REFERENCES Amos, L. A., Henderson, R. & Unwin, P. X. T. (1982). Proff. Biophy.s. Mol. Biol. 39, 183-231.

Beth c, H. A. (1933). In llandbuch der l'hysik (Geiger, H. & Schecl, K., eds), Band XXIV/I, IV Stosstheorie, i)p. 491-523, yon ,lulius Springer, Berlin. Chiu, W., Knapek, E., Jeng, T. W. & I)ietrich, I. (1981). Ultramicroscopy, 6, 291-296. l)owning, K. H. & Glaescr, R. M. (19S0). Biophys. J. 32, 851-856. Fujiyoshi, Y., Morikawa, K., Uyeda, N., Ozeki, H. & Yamagishi, H. (1983). Ullramlcroscopy. In the press. Fukami, A. & Adachi, K. (1965). J. El~:ctron .llicrosc. 14, 112-118. Howitt, D. G., Glaeser, R. M. & Thomas, ,1. (1976). J. Ulh'astruct. Ices. 55, 457-461. lsaacson, 31.8. (1973). In Physical Aspects of Electron Microscopy and Microbeam Analysis (Siegel, B. 31. & Bccman, I). R., eds), l)p. 247-258, John Wiley & Sons, New York. lsaaeson, M. 8. (1977). In Principles and Techniques of Electron .llicroscopy (Hayat, M. A., ed.), vol. 7, I)P. 1-78, Van Nostrand l{cinhold Co., Sew York. Isaacson, M. S., Johnson, 1). & Crew, A. V. (1973). Icadiat. ICes. 55,205-224. Ladner, J. E., l'inch, J. T., Klug, A. & Clark, B. F. C. (1972). J. Mol. Biol. 72, 99-101. 1,a(lner, J. E., ,lack, A., Robertis, J. I)., Brown, R. S., Rhodes, I)., Clark, B. F. C. & Klug, A. (1975). Proc. ,V~d. Acad. Sci., U.S.A. 72, 4414-4418. Lin, S. D. (1974). Radial. Ices. 59, 521-536. Unwin, P. N. T. & llenderson, R. (1975). J. Mol. Biol. 94, 425-440. Edited by A. Klug

1"Author to whom eorrespunden(.e should be addressed.