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On the negative staining of the protein crystal structure
Ultramicroscopy 7 ( 1981 ) 131 - 138 North-Holland Publishing Company
131
ON T H E NEGATIVE STAINING OF T H E P R O T E I N CRYSTAL STRUCTURE M.B. SHERMAN, E.V. ORLOVA, S.S. TERZYAN, R. K L E I N E * and N.A. KISELEV Institute of C~. stallography, Academy of Sciences of the USSR, 59 Leninsl~v Prospect, II 7333 Moscow, USSR Received 8 May 1981
It has been shown that in some cases negative staining reveals structure with details down to 0.4 n m in size, the nature of a protein playing a key role. Various stains seem to interact with different parts of a thermitase (a serine protease) molecule, which results in intensity changes in electron diffraction patterns.
I. Introduction The negative staining maps out the region of a specimen from which the stain is excluded. At the same time, the stain preserves the specimen structure during the process of drying. Obviously, the preservation of a dried stained specimen depends not only on the stain itself but also on the stability of the stain-specimen system and specimen nature. For instance, Unwin and Henderson have found that in purple membranes dried without preliminary treatment, the structural features are occasionally preserved, since these membranes are resistant to the action of various factors including dehydration [ 1]. The concept of negative staining is oversimplified since many stains (such as uranyl acetate) can interact with proteins. Unwin has observed an additional effect of positive staining during negative staining of catalase crystals [2]. It is noteworthy that the dose usually given to a specimen is ~ 1 C / c m 2, whereas - 10-3 C / c m 2 seems to be the maximum a protein molecule can tolerate. If an unstained dried protein is irradiated with a higher-than-critical dose of electrons, its structure is disordered [1]. A stained specimen als0 undergoes changes under irradiation with electrons
* Institute of Physiological Chemistry, Martin Luther University, Hollystrasse 1,402 Halle (Saale), GDR.
[3], while the stain is much more stable under irradiation. It is the presence of a stain which makes it possible to get a "cast" of a specimen with a resolution of 2 to 3 nm at doses of - 1 C / c m 2. It has been shown that electron diffraction is a very convenient technique for investigating ordered structures of thin protein crystals [4]. An electron diffraction pattern can be obtained at a considerably lower dose of irradiation as compared to that needed for producing images at a reasonable magnification of, say (3-5) × 10 4. The electron diffraction pattern permits verifying the crystallinity of the specimen. The further from the centre the visible diffraction spots, the more ordered the crystal is and the smaller the details of a reconstructed unit cell which can be revealed. With such a convenient method for checking the structure, one may also study the effect of preparation techniques on the initial specimen structure. Aqueous solutions of salts are "natural" media for biological specimens, and hence it is preferable to study them in water-saturated atmospheres. Indeed, as was shown in ref. [5], one can use an environmental cell to obtain the diffraction field up to 4 n m - I (e.g., for thin catalase crystals). Fast freezing also allows preservation of ordered crystals (e.g., catalase) [61. However, the described approaches are complicated in their realization. A somewhat simpler procedure has been proposed [1]: the crystals are immersed into a solution of
0304-3991/81/0000-0000/$02.50 © 1981 North-Holland
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M.B. Sherman et aL / Negatite ~taining of the protein ¢rvstal structure
small hydrophilic molecules, such as sugars, and then dried. A mixture of a sugar and a negative stain can also be used to facilitate observation of a "weak" electron diffraction pattern from a biological crystalline specimen [7]. In this case the intensity of low-order reflections is greatly increased, which brought the authors to the conclusion that the negative stain emphasizes mainly the coarse details while the sugar protects the fine structure of crystals from being disordered. It was assumed that a pure negative stain cannot preserve the small details detectable in X-ray analysis. As was found [2], the information gained from negatively stained catalase crystals is reliable at - 2 nm, while fine details of the structure are distorted, which is reminiscent of radiation damage. Nevertheless, we obtained electron diffraction patterns from thin crystals of a bacterial protease, thermitase, with diffraction field spread to - 4 nm- ~ (d = 0.25 nm) in the case when the crystals had been treated with glucose, and up to 2.5 nm L (d = 0.4 nm) in the case of conventional staining. In other words, sometimes negative staining in combination with the low-dose technique preserves the crystalline structure as if the crystal had been preliminarily treated with a sugar.
2. Materials and methods
Thermitase (TM) was isolated from culture medium of Thermoactinomices vulgaris by porous glass chromatography [8,9]. TM is a metal containing serine protease (two calcium atoms per TM molecule) and consists of one polipeptide chain comprising 305-+ 5 amino acid residues. The molecular weight of TM is 32,000-+ 1000 daltons [10,11 ]. TM microcrystals used for electron microscopy were prepared either by addition of ammonium sulphate to an aqueous solution of the enzyme (3 to 8 m g / m l ) up to final 1M concentration or by 10-15 rain evaporation of a droplet of the solution in air. This yields small plate-like enzyme crystals. The platelets are 20-60 nm thick and 1-20/~m wide. A suspension of microcrystals was applied to
grids covered with a thin carbon supporting film. Three kinds of preparation technique were used: (1) conventional negative staining [12] with a 1% uranyl acetate aqueous solution, or a 2% potassium silicotungstate solution, or a 2% phosphotungstic acid (PTA) solution; (2) staining with a mixture of a stain and a sugar, namely, either 0.5% uranyl acetate and 0.75% glucose or 1% PTA and 1% glucose; (3) "fixation" with glucose. Specimens were studied in a Philips EM 400 microscope at 100 kV, using the procedure described in detail in ref. [1]. Large plate-like TM crystals (0.25 X 0.25 × 0.05 mm s) were studied by X-ray analysis. Preliminary X-ray data were obtained for hkO and hOl zones in a precession camera (the crystal-to-film distance d = 75 ram, the precession angle 19°).
3. Results
Micrographs of negatively stained TM crystals with a resolution of - 2 nm as well as electron diffraction patterns were obtained from TM crystals prepared by all three techniques. Crystals irradiated during routine imaging at a magnification of 4 X 10 4 - 5 X 10 4 give electron diffraction patterns which reveal, if anything, only the spots corresponding to d = 2.4 nm. During this imaging a dose received by the crystals is of 0.1 1 C / c m 2 at magnification of 3 × 10 4 - 4 X 10 4. This value is much higher than the critical one ( - 10 3 C/cm2). Here, both the object and the stain can change. So the picture registered reveals radiation damages and not initial crystalline structure embedded in the amourphous stain matrix. Consequently, the images of crystals treated with different stains (figs. l a - l c ) appear to be alike and give similar optical Fourier spectra (figs. 2a-2c). In this case, the resolution is controlled by radiation resistance of a stain rather than by the crystallinity of an object. Fig. ld shows the image of a TM crystal "fixed" with glucose at a magnification of 3 X 10 4 but with a much lower dose than usually used: (2-5) X 10 _2 C / c m 2. According to the highest order diffraction spots visible in the optical Fourier transform (fig. 2d), the resolution in this case (fig. ld) is 0.9 nm. Thus, the resolution is better
M.B. Sherman et al. / Negative staining of the protein crystal structure
133
Fig. 1. Micrographs of ab projection of TM crystals. Magnification 4 × 105. Instrumental magnification: (a)-(c) 5 × 10a, (d) 3.9 × 104. The dose: (a)-(c) ~ 1 C/cm 2 and (d)~(2-5) × 10 2 C/cm 2. Staining with uranyl acetate (a), 2% potassium silicotungstate (b) and 2% PTA (c); (d) "fixed" with 1% glucose solution. The contrast in (d) is very low ("snap-shot" according to terminology of [14]).
and more information is available with the lower dose. Hitherto, electron diffraction patterns provided most of the data about TM crystals. The patterns shown in fig. 3 were taken with doses of 10 - 4 C / c m 2. It can be seen that the diffraction field was sufficiently large for all three types of crystal: "fixed" with glucose, with mixture of glucose and a stain, or negatively stained in a classical manner. However, if a suspension of TM crystals is dried in the absence of a stain or glucose, no peaks (even with small indices) can be discerned in the electron diffraction pattern. Thus, it seems that the stain or glucose stabilises the structure of TM in the process of drying, The diffraction field in the electron diffraction pattern shown in fig. 3a (crystal "fixed" with glucose) extends to 4 rim-J; in fact, the crystallin-
ity of the specimen is preserved even better: reflections of still higher order are visible in the patterns from TM crystals slightly tilted to the incident beam. It should be mentioned that the TM molecule does not contain carbohydrates linked by covalent bonds [11]. On the other hand, the purified enzyme displays strong affinity to mono- and polysaccharides, e.g. glucose or sucrose, or dextrins. This was proved by disc electrophoresis in the presence of sucrose and by gel filtration on Sephadex or Sepharose gels, respectively. In the first case only a TM protein zone originated from a TM-sucrose mixture contained neutral sugars; in the second case T M was desorbed in the presence of 1M NaCI [13]. The good tolerance of the TM crystal to drying in the presence of glucose can probably be explained by its strong affinity to
134
M.B. Sherman et al. / Negative staining of the protein co,stal structure
Fig. 2. Optical diffraction patterns from the images shown in fig. 1. The scale is indicated. Fig. 2d shows diffraction spots (encircled) corresponding to d=0.9 nm.
carbohydrates. And yet the resistance of structure to the process of drying in usual staining seems to be remarkable. Crystal lattice parameters and symmetry were determined by X-ray methods. The data show that the TM crystals are orthorhombic: a = 7.23 nm, b = 4.8 nm, c = 6.4 nm; space group P21212 ~, one T M molecule per asymmetric part of the unit cell. The X-ray diffraction field extends to 4 nm - I . Fig. 1 represents electron micrographs of the projection of stained crystals along the c axis; the ratio a / b (6/4.1 = 1.5) agrees with that obtained by the X-ray method. The absolute values of a and b could not be established from electron diffraction patterns with sufficient precision since the camera length was not fixed. The agreement of both a / b ratios suggests that the crystal order did
not change upon drying, i.e., that we deal with the native structure. When T M crystals are treated with a mixture of stain and glucose, the range of the diffraction field is also - 4 n m - J ; in the case of a pure stain, it is about 2.5 nm-~. Electron diffraction studies with low doses ( ~ 10 4 C//cm 2) prove that the diffraction field is large enough for all the types of treated crystals. Pattern symmetry is identical in all the types (P~). Nevertheless, using different stains and their mixtures with glucose, it is possible to obtain intensity changes for some diffraction peaks. For example, when PTA was taken, the peaks 200, 020, and 110 were found to be the most intensive; when T M crystals were "fixed" with glucose, a characteristic intensive "bracket" is seen formed by the spots with indices 16 0 0, 16 1 0, 15
M.B. Sherman et al. / Negative staining of the protein crystal structure
135
Fig. 3. Electron diffraction patterns from differently treated TM crystals (hkO plane) (a) "Fixed" with a I% glucose solution; the field spread up to 4 nm -I. (b) Mixture of uranyl acetate and glucose, or (c) mixture of PTA and glucose, or (d) 0.5% uranyl acetate, or (e) 1% PTA; (f) the same as (e) but with the shorter exposure. The field is ~ 2 . 5 n m - l in the case of (d) and (e).
136
M.B. Sherman et a L / Negattve staining of the protein ct3'stal structure
1 0 and by those symmetry-related to them. Figs. 3 a - 3 c shows that both "coarse" (reflections with small indices) and fine ( d ~ 0.45 nm) structure of the crystal are stain-dependent.
4. Discussion It should be emphasized that the result of protein-stain interaction depends on a stain. In other words, different stains will result in different images. The fact that actual images are similar indicates that the effect of high doses of irradiation on them is nearly the same, and the diffraction spots 200, 020 and 110 as well as those symmetry-related with them are very resistant to irradiation. This is confirmed by the fact that these spots are the most intensive in optical transforms (figs. 2a-2c). Such a dependence of intensities on a stain is similar to the situation occurring in X-ray search for isomorphous derivatives. It might happen that all the stained TM structures are isomorphous. We would like to note that the optical transforms shown in fig. 2 and the electron diffraction pattern in fig. 3f are similar. Therefore, the images in fig. 1 reveal the crude structure of the projection of T M crystal with a sufficient accuracy. Unwin's statement [2] seems to be valid for most investigations done hitherto: a negative stain maps out sufficiently well the structure of the particle only with the details down to ~ 2 nm in size. It is hardly possible to find a work using this method in which the resolution is essentially better than 2 n m . However it has been shown [7] that better results can be obtained with a stain "diluted" by glucose. These authors believe that glucose and not the stain was responsible for highangle reflections. Changes in the intensities of high-angle reflections in the case of T M indicate that the fine structure is also stain dependent. As was noted above, the stability of the stain-protein system is as important as the mere ability of the stain in preserving the structure during drying. As far as we know, T M is the only protein forming stable system with a stain, which does not dissociate upon dehydration and retains its regular structure with details down to 0.4 nm in size. Thus, one
may hope to gain valuable information about the T M molecule by using routine negative staining. We should like to emphasize that the intensities of the reflexes in electron diffraction patterns depend on the stain. This phenomenon can facilitate the determination of the structure factor phases from comparison of patterns in a way similar to that in X-ray diffraction analysis. H o p p e [15] indicates that such a method of phase determination has one but serious disadvantage: the electron microscope is not used as a system able to register both amplitudes and phases of structure factors. Yet the method enables relatively simple registration of electron diffraction patterns under low irradiation conditions.
Acknowledgments We would like to thank professor B.K. Vainstein for interest in our work and helpful discussion.
References [I] P.N.T. Unwin and R. Henderson, J. Mol. Biol. 94 (1975) 425. [2] P.N.T. Unwin, J. Mol. Biol. 98 (1975) 235. [3] P.N.T. Unwin, Proc. Roy. Soc. (London) A329 (1977) 327. [4] R.M. Glaeser and G. Thomas, Biophys. J. 9 (1969) 1073. [5] V.R. Matricardi, R.C. Moretz and D.F. Parsons, Science 177 (1972) 268. [6] K.A. Taylor and R.M. Glaeser, in: Proc. 8th Intern. Congr. on Electron Microscopy, Canberra, 1974, Vol. II, Eds. Sanders and Goodchild (Australian Acad. Sci.. Canberra) pp. 64-65. [7] W. Chiu and I. Hosoda, in: Proc. 9th Intern. Congr. on Electron Microscopy, Toronto, 1978, Vol. II, Ed. J.M. Sturgess (Microscopical Soc. Canada, Toronto) pp. 178179. [8] R. Kleine, U. Rothe, U, Kettmann and H, Schelle, Lecture at Intern. Syrup. on Proteinases and their Inhibitors, Portoroz, Yugoslavia, 1980 (Pergamon, 1981 ). [9] R. Kleine, U. Rothe, H. Rfittloff and U. Behnke, G D R Wirtchafts-Patent [978 WP C07 G/210135. [10] G. Hausdorf, K. Kri~ger and W.E. H6hne, Intern. J. Peptide Proteinase Res. 15 (1980) 420. [11] U. Kettmann, U. Rothe, I. Marqt~ardt and R. Kleine, 12th FEBS Meeting, Dresden, 1978. [12] H.E. Huxley and G. Zubay, J. Mol. Biol. 2 (1960) 10.
M.B. Sherman et al. / Negative staining of the protein crystal structure [13] R. Kleine, H. Janeke and S. Fensterseiter, 1st Joint Symp. GDR-(~SSR on Enzyme Applications, Reinhardsbri~nn, GDR, 1980. [14] I.A.M. Kuo and R.M. Glaeser, Ultramicroscopy I (1975) 53.
137
[15] W. Hoppe, Three-Dimensional Low Dose Reconstruction of Periodical Aggregates, in: Unconventional Electron Microscopy for Molecular Structure Determination, Eds. W. Hoppe and R. Mason (Vieweg, Braunschweig, 1979) p. 191.
131
ON T H E NEGATIVE STAINING OF T H E P R O T E I N CRYSTAL STRUCTURE M.B. SHERMAN, E.V. ORLOVA, S.S. TERZYAN, R. K L E I N E * and N.A. KISELEV Institute of C~. stallography, Academy of Sciences of the USSR, 59 Leninsl~v Prospect, II 7333 Moscow, USSR Received 8 May 1981
It has been shown that in some cases negative staining reveals structure with details down to 0.4 n m in size, the nature of a protein playing a key role. Various stains seem to interact with different parts of a thermitase (a serine protease) molecule, which results in intensity changes in electron diffraction patterns.
I. Introduction The negative staining maps out the region of a specimen from which the stain is excluded. At the same time, the stain preserves the specimen structure during the process of drying. Obviously, the preservation of a dried stained specimen depends not only on the stain itself but also on the stability of the stain-specimen system and specimen nature. For instance, Unwin and Henderson have found that in purple membranes dried without preliminary treatment, the structural features are occasionally preserved, since these membranes are resistant to the action of various factors including dehydration [ 1]. The concept of negative staining is oversimplified since many stains (such as uranyl acetate) can interact with proteins. Unwin has observed an additional effect of positive staining during negative staining of catalase crystals [2]. It is noteworthy that the dose usually given to a specimen is ~ 1 C / c m 2, whereas - 10-3 C / c m 2 seems to be the maximum a protein molecule can tolerate. If an unstained dried protein is irradiated with a higher-than-critical dose of electrons, its structure is disordered [1]. A stained specimen als0 undergoes changes under irradiation with electrons
* Institute of Physiological Chemistry, Martin Luther University, Hollystrasse 1,402 Halle (Saale), GDR.
[3], while the stain is much more stable under irradiation. It is the presence of a stain which makes it possible to get a "cast" of a specimen with a resolution of 2 to 3 nm at doses of - 1 C / c m 2. It has been shown that electron diffraction is a very convenient technique for investigating ordered structures of thin protein crystals [4]. An electron diffraction pattern can be obtained at a considerably lower dose of irradiation as compared to that needed for producing images at a reasonable magnification of, say (3-5) × 10 4. The electron diffraction pattern permits verifying the crystallinity of the specimen. The further from the centre the visible diffraction spots, the more ordered the crystal is and the smaller the details of a reconstructed unit cell which can be revealed. With such a convenient method for checking the structure, one may also study the effect of preparation techniques on the initial specimen structure. Aqueous solutions of salts are "natural" media for biological specimens, and hence it is preferable to study them in water-saturated atmospheres. Indeed, as was shown in ref. [5], one can use an environmental cell to obtain the diffraction field up to 4 n m - I (e.g., for thin catalase crystals). Fast freezing also allows preservation of ordered crystals (e.g., catalase) [61. However, the described approaches are complicated in their realization. A somewhat simpler procedure has been proposed [1]: the crystals are immersed into a solution of
0304-3991/81/0000-0000/$02.50 © 1981 North-Holland
132
M.B. Sherman et aL / Negatite ~taining of the protein ¢rvstal structure
small hydrophilic molecules, such as sugars, and then dried. A mixture of a sugar and a negative stain can also be used to facilitate observation of a "weak" electron diffraction pattern from a biological crystalline specimen [7]. In this case the intensity of low-order reflections is greatly increased, which brought the authors to the conclusion that the negative stain emphasizes mainly the coarse details while the sugar protects the fine structure of crystals from being disordered. It was assumed that a pure negative stain cannot preserve the small details detectable in X-ray analysis. As was found [2], the information gained from negatively stained catalase crystals is reliable at - 2 nm, while fine details of the structure are distorted, which is reminiscent of radiation damage. Nevertheless, we obtained electron diffraction patterns from thin crystals of a bacterial protease, thermitase, with diffraction field spread to - 4 nm- ~ (d = 0.25 nm) in the case when the crystals had been treated with glucose, and up to 2.5 nm L (d = 0.4 nm) in the case of conventional staining. In other words, sometimes negative staining in combination with the low-dose technique preserves the crystalline structure as if the crystal had been preliminarily treated with a sugar.
2. Materials and methods
Thermitase (TM) was isolated from culture medium of Thermoactinomices vulgaris by porous glass chromatography [8,9]. TM is a metal containing serine protease (two calcium atoms per TM molecule) and consists of one polipeptide chain comprising 305-+ 5 amino acid residues. The molecular weight of TM is 32,000-+ 1000 daltons [10,11 ]. TM microcrystals used for electron microscopy were prepared either by addition of ammonium sulphate to an aqueous solution of the enzyme (3 to 8 m g / m l ) up to final 1M concentration or by 10-15 rain evaporation of a droplet of the solution in air. This yields small plate-like enzyme crystals. The platelets are 20-60 nm thick and 1-20/~m wide. A suspension of microcrystals was applied to
grids covered with a thin carbon supporting film. Three kinds of preparation technique were used: (1) conventional negative staining [12] with a 1% uranyl acetate aqueous solution, or a 2% potassium silicotungstate solution, or a 2% phosphotungstic acid (PTA) solution; (2) staining with a mixture of a stain and a sugar, namely, either 0.5% uranyl acetate and 0.75% glucose or 1% PTA and 1% glucose; (3) "fixation" with glucose. Specimens were studied in a Philips EM 400 microscope at 100 kV, using the procedure described in detail in ref. [1]. Large plate-like TM crystals (0.25 X 0.25 × 0.05 mm s) were studied by X-ray analysis. Preliminary X-ray data were obtained for hkO and hOl zones in a precession camera (the crystal-to-film distance d = 75 ram, the precession angle 19°).
3. Results
Micrographs of negatively stained TM crystals with a resolution of - 2 nm as well as electron diffraction patterns were obtained from TM crystals prepared by all three techniques. Crystals irradiated during routine imaging at a magnification of 4 X 10 4 - 5 X 10 4 give electron diffraction patterns which reveal, if anything, only the spots corresponding to d = 2.4 nm. During this imaging a dose received by the crystals is of 0.1 1 C / c m 2 at magnification of 3 × 10 4 - 4 X 10 4. This value is much higher than the critical one ( - 10 3 C/cm2). Here, both the object and the stain can change. So the picture registered reveals radiation damages and not initial crystalline structure embedded in the amourphous stain matrix. Consequently, the images of crystals treated with different stains (figs. l a - l c ) appear to be alike and give similar optical Fourier spectra (figs. 2a-2c). In this case, the resolution is controlled by radiation resistance of a stain rather than by the crystallinity of an object. Fig. ld shows the image of a TM crystal "fixed" with glucose at a magnification of 3 X 10 4 but with a much lower dose than usually used: (2-5) X 10 _2 C / c m 2. According to the highest order diffraction spots visible in the optical Fourier transform (fig. 2d), the resolution in this case (fig. ld) is 0.9 nm. Thus, the resolution is better
M.B. Sherman et al. / Negative staining of the protein crystal structure
133
Fig. 1. Micrographs of ab projection of TM crystals. Magnification 4 × 105. Instrumental magnification: (a)-(c) 5 × 10a, (d) 3.9 × 104. The dose: (a)-(c) ~ 1 C/cm 2 and (d)~(2-5) × 10 2 C/cm 2. Staining with uranyl acetate (a), 2% potassium silicotungstate (b) and 2% PTA (c); (d) "fixed" with 1% glucose solution. The contrast in (d) is very low ("snap-shot" according to terminology of [14]).
and more information is available with the lower dose. Hitherto, electron diffraction patterns provided most of the data about TM crystals. The patterns shown in fig. 3 were taken with doses of 10 - 4 C / c m 2. It can be seen that the diffraction field was sufficiently large for all three types of crystal: "fixed" with glucose, with mixture of glucose and a stain, or negatively stained in a classical manner. However, if a suspension of TM crystals is dried in the absence of a stain or glucose, no peaks (even with small indices) can be discerned in the electron diffraction pattern. Thus, it seems that the stain or glucose stabilises the structure of TM in the process of drying, The diffraction field in the electron diffraction pattern shown in fig. 3a (crystal "fixed" with glucose) extends to 4 rim-J; in fact, the crystallin-
ity of the specimen is preserved even better: reflections of still higher order are visible in the patterns from TM crystals slightly tilted to the incident beam. It should be mentioned that the TM molecule does not contain carbohydrates linked by covalent bonds [11]. On the other hand, the purified enzyme displays strong affinity to mono- and polysaccharides, e.g. glucose or sucrose, or dextrins. This was proved by disc electrophoresis in the presence of sucrose and by gel filtration on Sephadex or Sepharose gels, respectively. In the first case only a TM protein zone originated from a TM-sucrose mixture contained neutral sugars; in the second case T M was desorbed in the presence of 1M NaCI [13]. The good tolerance of the TM crystal to drying in the presence of glucose can probably be explained by its strong affinity to
134
M.B. Sherman et al. / Negative staining of the protein co,stal structure
Fig. 2. Optical diffraction patterns from the images shown in fig. 1. The scale is indicated. Fig. 2d shows diffraction spots (encircled) corresponding to d=0.9 nm.
carbohydrates. And yet the resistance of structure to the process of drying in usual staining seems to be remarkable. Crystal lattice parameters and symmetry were determined by X-ray methods. The data show that the TM crystals are orthorhombic: a = 7.23 nm, b = 4.8 nm, c = 6.4 nm; space group P21212 ~, one T M molecule per asymmetric part of the unit cell. The X-ray diffraction field extends to 4 nm - I . Fig. 1 represents electron micrographs of the projection of stained crystals along the c axis; the ratio a / b (6/4.1 = 1.5) agrees with that obtained by the X-ray method. The absolute values of a and b could not be established from electron diffraction patterns with sufficient precision since the camera length was not fixed. The agreement of both a / b ratios suggests that the crystal order did
not change upon drying, i.e., that we deal with the native structure. When T M crystals are treated with a mixture of stain and glucose, the range of the diffraction field is also - 4 n m - J ; in the case of a pure stain, it is about 2.5 nm-~. Electron diffraction studies with low doses ( ~ 10 4 C//cm 2) prove that the diffraction field is large enough for all the types of treated crystals. Pattern symmetry is identical in all the types (P~). Nevertheless, using different stains and their mixtures with glucose, it is possible to obtain intensity changes for some diffraction peaks. For example, when PTA was taken, the peaks 200, 020, and 110 were found to be the most intensive; when T M crystals were "fixed" with glucose, a characteristic intensive "bracket" is seen formed by the spots with indices 16 0 0, 16 1 0, 15
M.B. Sherman et al. / Negative staining of the protein crystal structure
135
Fig. 3. Electron diffraction patterns from differently treated TM crystals (hkO plane) (a) "Fixed" with a I% glucose solution; the field spread up to 4 nm -I. (b) Mixture of uranyl acetate and glucose, or (c) mixture of PTA and glucose, or (d) 0.5% uranyl acetate, or (e) 1% PTA; (f) the same as (e) but with the shorter exposure. The field is ~ 2 . 5 n m - l in the case of (d) and (e).
136
M.B. Sherman et a L / Negattve staining of the protein ct3'stal structure
1 0 and by those symmetry-related to them. Figs. 3 a - 3 c shows that both "coarse" (reflections with small indices) and fine ( d ~ 0.45 nm) structure of the crystal are stain-dependent.
4. Discussion It should be emphasized that the result of protein-stain interaction depends on a stain. In other words, different stains will result in different images. The fact that actual images are similar indicates that the effect of high doses of irradiation on them is nearly the same, and the diffraction spots 200, 020 and 110 as well as those symmetry-related with them are very resistant to irradiation. This is confirmed by the fact that these spots are the most intensive in optical transforms (figs. 2a-2c). Such a dependence of intensities on a stain is similar to the situation occurring in X-ray search for isomorphous derivatives. It might happen that all the stained TM structures are isomorphous. We would like to note that the optical transforms shown in fig. 2 and the electron diffraction pattern in fig. 3f are similar. Therefore, the images in fig. 1 reveal the crude structure of the projection of T M crystal with a sufficient accuracy. Unwin's statement [2] seems to be valid for most investigations done hitherto: a negative stain maps out sufficiently well the structure of the particle only with the details down to ~ 2 nm in size. It is hardly possible to find a work using this method in which the resolution is essentially better than 2 n m . However it has been shown [7] that better results can be obtained with a stain "diluted" by glucose. These authors believe that glucose and not the stain was responsible for highangle reflections. Changes in the intensities of high-angle reflections in the case of T M indicate that the fine structure is also stain dependent. As was noted above, the stability of the stain-protein system is as important as the mere ability of the stain in preserving the structure during drying. As far as we know, T M is the only protein forming stable system with a stain, which does not dissociate upon dehydration and retains its regular structure with details down to 0.4 nm in size. Thus, one
may hope to gain valuable information about the T M molecule by using routine negative staining. We should like to emphasize that the intensities of the reflexes in electron diffraction patterns depend on the stain. This phenomenon can facilitate the determination of the structure factor phases from comparison of patterns in a way similar to that in X-ray diffraction analysis. H o p p e [15] indicates that such a method of phase determination has one but serious disadvantage: the electron microscope is not used as a system able to register both amplitudes and phases of structure factors. Yet the method enables relatively simple registration of electron diffraction patterns under low irradiation conditions.
Acknowledgments We would like to thank professor B.K. Vainstein for interest in our work and helpful discussion.
References [I] P.N.T. Unwin and R. Henderson, J. Mol. Biol. 94 (1975) 425. [2] P.N.T. Unwin, J. Mol. Biol. 98 (1975) 235. [3] P.N.T. Unwin, Proc. Roy. Soc. (London) A329 (1977) 327. [4] R.M. Glaeser and G. Thomas, Biophys. J. 9 (1969) 1073. [5] V.R. Matricardi, R.C. Moretz and D.F. Parsons, Science 177 (1972) 268. [6] K.A. Taylor and R.M. Glaeser, in: Proc. 8th Intern. Congr. on Electron Microscopy, Canberra, 1974, Vol. II, Eds. Sanders and Goodchild (Australian Acad. Sci.. Canberra) pp. 64-65. [7] W. Chiu and I. Hosoda, in: Proc. 9th Intern. Congr. on Electron Microscopy, Toronto, 1978, Vol. II, Ed. J.M. Sturgess (Microscopical Soc. Canada, Toronto) pp. 178179. [8] R. Kleine, U. Rothe, U, Kettmann and H, Schelle, Lecture at Intern. Syrup. on Proteinases and their Inhibitors, Portoroz, Yugoslavia, 1980 (Pergamon, 1981 ). [9] R. Kleine, U. Rothe, H. Rfittloff and U. Behnke, G D R Wirtchafts-Patent [978 WP C07 G/210135. [10] G. Hausdorf, K. Kri~ger and W.E. H6hne, Intern. J. Peptide Proteinase Res. 15 (1980) 420. [11] U. Kettmann, U. Rothe, I. Marqt~ardt and R. Kleine, 12th FEBS Meeting, Dresden, 1978. [12] H.E. Huxley and G. Zubay, J. Mol. Biol. 2 (1960) 10.
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