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Electron microtephroscopy of proteins. A close look at the ashes of myokinase and protamine
JOURNAL OF ULTRASTRUCTURERESEARCH 52, 1 9 3 - 2 0 1
(1975)
Electron M i c r o t e p h r o s c o p y of Proteins A Close Look at the Ashes of Myokinase and Protamine F. P. OTTENSMEYER, R. F. WHITING, E. E. SCHMIDT,1 AND R. S. CLEMENS Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Canada M 4 X 1K9 Received September 27, 1974 The enormous radiation doses required for the observation of biological specimens in the electron microscope suggest t h a t the observation of unstained unshadowed macromolecules should be called microtephroscopy, the study of ashes with the microscope. In this light, our observation of fine-structure well below 10 A in the dark-field electron micrographs of myokinase and protamine becomes an exciting starting point from which to attempt the reconstruction of the biological structure of macromolecules at such a resolution. Moreover, the repeated observation of virtually identical images, as well as correlation with structure determined by X-ray crystallography argues that much of the detail observed is still biologically meaningful structure. INTRODUCTION
The examination of biological specimens in the electron microscope inevitably subjects them to enormous radiation doses. Isaacson et al. (4) have estimated that even with minimal irradiation techniques a dose of 100 electrons/A 2, or about 1011 rads, is required to realize a resolution of about 10 /k in the image. With such a dose one would assume that very little meaningful information at any level, not to speak of 10 Jr, is retained. Their measurements, using energy-loss spectra and electron diffraction, indicate that within a factor of 10 of such doses a large number of biological substances retain on the average about a third of their structural integrity (4). This result can be interpreted in several ways. Pessimistically, one can say most of the biological specimen is destroyed. Optimistically, however, some of the specimen is still intact. Moreover, if one supposes that loss of native electronic structure does not necessarily mean loss of all material, nor that loss of three-dimensional order means loss of two-dimensional projected detail, then an interesting possibility remains. Let the biological structure under the intense ra1Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
diation crumble into a heap of carbon, nitrogen, and oxygen atoms. In some way this heap of atomic ashes must bear a relationship to the specimen from which it was produced. The question that must be answered is how tenuous this relationship is, or to what resolution a biologically significant structural resemblance is maintained. In recognition of the fact that we are only perceiving the crumbling remains of the biological specimen, we have chosen to call this study microtephroscopy, a close look at the ashes. The results presented here indicate that with care, dark-field images of unstained and unshadowed proteins exhibit structural detail that is well below 10 A. Moreover, in one instance, x-ray crystallographic structure determination shows that much of this detail is biologically meaningful structure. Early work in dark field (5) had already indicated that helices in DNA, loops in tRNA, and the cleft in ribonuclease could be seen. It could be argued, however, that prior knowledge of such well-studied structures introduced a degree of subjectivity into the search for images resembling the desired configurations. Because the destruction wrought by the radiation dose mentioned above prevented the observa193
Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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t i o n of large fields of w e l l - p r e s e r v e d i d e n t i cal s p e c i m e n s , s u c h a r g u m e n t s c o u l d n o t be c o u n t e r e d easily. S e v e r a l years ago to d i s p e l s u c h d o u b t s in our l a b o r a t o r y a n d to test our consistency in arriving at struct u r e s , we i n v e s t i g a t e d d a r k - f i e l d i m a g e s of m y o k i n a s e , a p r o t e i n whose s t r u c t u r e was unknown at that time. Subsequent x-ray a n a l y s i s a t low r e s o l u t i o n (6) r e a s s u r e d u s of t h e c o r r e c t n e s s of o u r gross s t r u c t u r a l a s s i g n m e n t . I n a d d i t i o n , however, reexa m i n a t i o n a n d c o m p a r i s o n of our i m a g e s w i t h r e c e n t h i g h r e s o l u t i o n x - r a y d a t a (7) often indicated a structural identity at levels b e t w e e n 6 a n d 10 /~. W e h a d origin a l l y d i s r e g a r d e d s u c h fine s t r u c t u r e as b e i n g m e a n i n g l e s s i n l i g h t of t h e r a d i a t i o n burden. T h e s e r e t r o s p e c t i v e d a t a on m y o k i n a s e are d e t a i l e d i n t h i s p a p e r . H o w e v e r , elect r o n m i c r o g r a p h s of a s e c o n d p r o t e i n , herr i n g s p e r m p r o t a m i n e , are s h o w n as well. T h e s e i m a g e s a g a i n show r e p r o d u c i b l e rep e a t e d d e t a i l a t a r e s o l u t i o n of 5-10 ~k. For this protein no independent confirmation of t h e s t r u c t u r e h a s b e e n c a r r i e d o u t as yet. METHODS AND MATERIALS Myokinase (pig muscle, Sigma Chemical Co., St. Louis, Mo.) was used at 0.1-10 #g/ml in 0.2 M ammonium acetate. Herring protamine sulphate (Mann Research Laboratories, New York) was separated into fractions Y-II, Y-I, and Z (3) and was desalted by loading on a CM-eellulose column in the protonated form, washing with distilled water, and eluting with 0.2 M HC1. The fractions were lyophilized and redissolved in distilled water for electron microscopy. Myokinase was examined on a Philips EM300 electron microscope nominally at 51 300X, protamine on a Siemens Elmiskop 102 at 50 000 and 100 000X. Magnifications were calibrated and corrected using a grating replica (Fullam, Schenectady, N. Y.). Both specimens were supported on thin carbon films ( < 20 ~) on fenestrated plastic supports (8). To obtain the minimum possible exposure a technique was followed that differed slightly for each of the two instruments. On the EM300 the beam at cross-over on the screen was carefully centered mechanically with the deflection system off. The deflection system was then reengaged and centered as well. With the beam still condensed to cover only the viewing screen so that no extraneous region would
receive an exposure, the microscope was now focused on an area of the specimen adjacent to the one to be photographed. The beam was electronically deflected to the edge of viewing screen so that only a mere sliver of the periphery of the screen was illuminated as an aid for the next step. The unirradiated specimen area was now moved into the center, a photo plate was transported under the screen, and the screen was lifted. Neither the desired specimen area nor the photographic plate were exposed at this point, since the beam was deflected off the field of view. After a few seconds, to permit mechanical vibrations to damp out, the exposure button was pressed while at the same time the electronic deflection system was shut off. This returned the beam to the mechanically aligned central position exposing the specimen and the plate. After the preset exposure time the shutter engaged and the plate was transported out of the way. On the Elmiskop 102 a second and independent control on the electronic deflection system was installed. Here the first control served to align the beam initially. The second control, which could be disengaged, then was used in a manner analogous to the deflection system of the EM300 above. RESULTS
M i n i m u m beam exposure. O u r t e c h n i q u e of m i n i m i z i n g e x p o s u r e to t h e specimen initially produced extremely frustrati n g r e s u l t s . S i n c e o n l y t h e a r e a a d j a c e n t to t h e one p h o t o g r a p h e d was u s e d to focus, we e x p e c t e d s o m e p l a t e s to be o u t of focus. A c h e c k o n t h e v i e w i n g s c r e e n after t h e i m a g e was r e c o r d e d r e v e a l e d t h a t a b o u t t h r e e o u t of four i m a g e s h a d b e e n f o c u s e d correctly. S u r p r i s i n g l y , however, a large n u m b e r of p l a t e s i n d i c a t e d m o v e m e n t or s t r e a k i n g of t h e i m a g e , s o m e t i m e s i n one d i r e c t i o n , t h e n at another angle on the next plate, s o m e t i m e s f a n - s h a p e d as t h o u g h e m a n a t ing f r o m s o m e p o i n t off the p l a t e . C a r e f u l e x a m i n a t i o n of " g o o d " p l a t e s i n d i c a t e d that even these exhibited streaking radially a w a y f r o m a s t i g m a t i c i n - f o c u s r e g i o n i n t h e c e n t r a l p o r t i o n of t h e b e a m . Fig. 1 shows a n e x a m p l e of t h i s p h e n o m e n o n . If a s e c o n d e x p o s u r e is m a d e of t h e i d e n t i c a l area, l e a v i n g t h e b e a m on t h e s p e c i m e n , the second micrograph appears perfectly n o r m a l , s h o w i n g n o n e of t h e effect. W e c a n o n l y s u g g e s t t h a t w h a t we o b s e r v e is t h e t r a n s i e n t t h a t occurs as t h e u n e x p o s e d cold u n c h a r g e d region heats up a n d charges
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195
FIo. 1. Typical appearance of radial streaking observed under our minimum exposure conditions in dark field. The center of the beam is revealed by the usefuI area of the micrograph slightly below and left of center. The specimen is myokinase on a thin carbon support, x 79 000. under the action of the beam. Once equilibrium is reached, the image remains steady. U n f o r t u n a t e l y by this time the specimen has received more t h a n a minimal dose, resulting in disappearance of fine detail in the structure, often beyond.a 20 A resolution, r e a r r a n g e m e n t of mass, and the a p p e a r a n c e of intense bright spots. Therefore, to preserve as m u c h fine structure as possible we have accepted the smaller useful field of view in the central portion of the b e a m and the need for careful centering of this b e a m on the camera of the microscope. Myokinase. T o obtain a measure of our consistency in determining unknown structures of macromolecules three of us two years ago i n d e p e n d e n t l y scanned five original plates of images of myokinase. These micrographs had been taken using different concentrations of the molecule and had been checked to assure that they were
in focus. The sole known facts were the molecular weight of the protein (22 000) and the magnification of the plates. Our aim was to ascertain independently the shape of the molecule, with about 25 to 30 examples each, m a r k e d on the plates to back up the structural assignment. All three of us arrived at a C-shaped molecule about 50 A in size, with one arm of the C slightly less dense than the other. Moreover, although we had looked at the same plates, none of us had chosen the same molecules. The structures picked generally were well isolated from one another, and, though of low contrast, were significantly different from the clean though motley carbon support film. An example of an area containing a p u r p o r t e d myokinase structure is shown in Fig. 2. This grayish white structure is distinct in shape, size, and contrast from background. However, only repeated observation of this type of struc-
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FIG. 2. Large area dark field image containing a single molecule of myokinase (arrow). The shape and contrast are distinctly different from background mottle. × 1 100 000.
ture as well as of the r a n d o m mottle of e m p t y carbon films can instil such confidence. This and other examples at higher magnification are shown in Fig. 3. These images are shown in reverse contrast since an intermediate enlargement was used to achieve this magnification. The C-shape generally predominates, though dumbbell structures (possibly a different projected view) as well as nondescript shapes are also found (bottom row, Fig. 3). We accepted a fairly noncontroversial image resolution of about 20 A at this stage and were pleased when low resolution x-ray diffraction studies suggested t h a t our assignment of the size and C-shape to myokinase was not only consistent b u t correct
(6). Recent x-ray studies at 3 A (7) made us
reexamine our work. M a n y of our images had a distinct loop or appendage on one side or the other near the b o t t o m (Fig. 3 ) . In addition, there was the occasional loop in the arms of the C-shape. One very striking example is enlarged in Fig. 4a alongside the drawing of the polypeptide chain of the molecule d e t e r m i n e d from x-ray analysis. If one considers t h a t the outside dimension of the molecule is 50 A then the observed structure in the image exhibits a resolution of 10 A or better. Moreover, since comparison with the x-ray d e t e r m i n a t i o n indicates a striking similarity, one is t e m p t e d to conclude t h a t the observed structure corresponds to biologically meaningful detail. P r o t a m i n e . T h e smallest proteins we have e x a m i n e d to date are the herring
197
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Fla. 3, Examples of shapes observed on dark field micrographs of myokinase. Reverse contrast, x 2 600 000.
@ 4b FIG. 4. Comparison of myokinase structure observed in dark field with the structure determined by X-ray crystallography [taken from (6) with permission]. × 4 200 000.
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sperm protamines. They have a molecular weight of about 4100 and have 20 or 21 arginine residues out of their 30 or 31 total amino acids. Their primary sequence is known but their structure has not yet been determined (1, 2). Our dark field micrographs indicate that the unstained unshadowed molecules are basically ellipsoidal in shape about 25 A in the shorter dimension and 35-40 A in the longer. Closer examination, however, reveals a substructure that is fan-shaped, comb-shaped, or star-shaped, often with six major leaves, tines, or rays. One such example is shown in the large area view in Fig. 5. It is possible that other prominent light areas in the figure may represent further molecules. However, these were too nondescript, possibly too
degraded, to merit further consideration, or their general shape and structure was not found repeatedly in other areas. Reproducibility and consistency in structure in a size range that could correspond to a molecular weight of 4100 were virtually the only criteria we could apply in this structure determination. For sizes larger or smaller than the above dimensions no consistent structures could be found at all. Within that size range, however, the task was relatively easy. Fig. 6a shows examples found in fraction Y-II, Fig. 6b molecules from fraction Y-I, and Fig. 6c from fraction Z. As for Figs. 3 and 4, these high magnification images are shown in reverse contrast. Although we have not made an exhaustive study, there appears to be little difference in structure between
FIG. 5. Large area dark field micrograph of a spread of protamine molecules. One image (marked by the arrow) is clearly characteristically different from random background. Other spots are rich enough in contrast to be distinct from background, but do not show enough detail to be useful for structure determination. × 1 100 000.
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199
FIG. 6. Examples of structurally interesting shapes observed in dark field micrographs of herring sperm protamine: (a) fraetion Y-II; (b) fraction Y-I; (c) fraction Z. Reverse contrast. × 3 500 000. the fractions. T h e similarity on the other h a n d is m u c h more striking. The structures are not all identical. Nevertheless, they can be easily related, assuming a small a m o u n t of flexibility in the molecule.
While here we c a n n o t yet claim fine s t r u c t u r e with biological significance, whatever has r e m a i n e d of the p r o t a m i n e molecule exhibits a reproducible structure with detail frequently at the 5 A level.
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Two examples, which are virtually identical in every detail, are shown in Fig. 7. For ease of m e a s u r e m e n t the scale here is 2 A/mm. These molecules came from different fractions on the gradient (though both are probably Y-I) and were taken at different electron optical magnifications (Fig. 7a at × 100 000, Fig. 7b at × 50 000). Since the radiation burden for the two images differed approximately by a factor of four, the similarity in structure is all the more remarkable. Nor are they isolated examples. The first image in Fig. 6 could almost be added to this pair, while several other structures in Fig. 6, though different from these, resemble each other quite closely. DISCUSSION We have e x a m i n e d dark field images of two proteins in an a t t e m p t to decide if meaningful structure d e t e r m i n a t i o n with the electron microscope at a resolution level of b e t t e r t h a n 20 A is possible. Our micrographs indicate t h a t reproducible detail is visible even at a 5 A level in m a n y images. Since comparison with x-ray d a t a has indicated for myokinase t h a t at least some of this detail represents biological structure, why should some of the detail in the images of p r o t a m i n e not also be biologically significant? If the electron b o m b a r d m e n t forms nonvolatile radicals or excited states in the macromolecule, the molecule can either do nothing, or react internally or with the carbon support. Gross rearrange-
ments of structure very likely would not be reproduced consistently. Such configurations would be discarded in the search for repeatedly similar structures. Minor alterations such as reactions with a neighboring amino acid side chain, even cross-linking with a close peptide chain in the same molecule would not necessarily be readily recognizable; nor would reactions with an adjacent portion of the carbon support film. Such a reaction might even stabilize the macromolecule a m o m e n t longer. As long as the two-dimensional projection of the mass of the molecule remains relatively constant, whatever the vertical rearrangem e n t might be, the electron micrograph would present a relatively faithful reproduction. Moreover, the repeated observation of virtually identical fine structure well below 10 A argues t h a t in these instances the structural alteration is probably minor, rather t h a n t h a t c o m m o n gross structural modifications have resulted in an identical end product. Our bias is obvious. We propose t h a t the finger-like segments of the images of protamine, often separated by as little as 5 A, are biologically significant. Proof of this on the one hand m u s t wait for x-ray crystallographic data. On the other hand, further electron microscopic studies of a modified protamine, the phosphorylated species, or a molecule m a r k e d with a specific localized cluster of heavy atoms m a y also shed some light on the verity of our proposal. T h e latter approach has already been shown to
FIG. 7. Two dark field micrographs of protamine that are virtually identical in every detail showing structure with measurable 5/~ image resolution: (a) from fraction Y-I, taken at × 100 000; (b) from fraction Y-I, taken at × 50 000. Since the final magnification is the same, the difference in electron optical magnification accounts for the difference in granularity of the images. × 5 000 000.
MICROTEPHROSCOPY OF PROTEINS
be feasible in the case of transfer RNA (Korn and Ottensmeyer, in preparation). Finally, however, we think that our results have shown that too pessimistic an approach to the resolution problem is not warranted. A careful examination of the images of even crumbling biological structures may reveal much about their original high resolution detail. This work was supported by the National Cancer Institute of Canada and grant MA3763 of the National Research Council of Canada. The second author held an NCI of Canada Fellowship. Figure 4b was kindly provided by Drs. G. E. Schulz and R. H. Schirmer.
201
REFERENCES 1. ANDO,T., ANDSUZUKI,K., Biochim. Biophys. Acta 121, 427 (1966). 2. ANDO,T., ANDSUZUKI,K., Biochim. Biophys. Acta 140, 375 (1967). 3. ANDO, T., AND WATANABE,S., Internat. J. Protein Res. l, 221 (1969). 4. ISAACSON, M., JOHNSON, D., AND CREWE, A. V., Radiat. Res. 55, 205 (1973). 5. OTTENSMEYER,f. P., Biophys. J. 9, 1144 (1969). 6. SCHULZ,G. E., BIEDERMANN,K., KABSCH,W., AND SCHIRMER, R. H., J. Mol. Biol. 80, 857 (1973). 7. SCHULZ,G. E., ELZINGA,M., MARX, F., ANDSCHIRMER, R. H., Nature 250, 120 (1974). 8. WHITING, R. F., AND OTTENSMEYER,F. P., J. Mol. Biol. 67, 173 (1972).
(1975)
Electron M i c r o t e p h r o s c o p y of Proteins A Close Look at the Ashes of Myokinase and Protamine F. P. OTTENSMEYER, R. F. WHITING, E. E. SCHMIDT,1 AND R. S. CLEMENS Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Canada M 4 X 1K9 Received September 27, 1974 The enormous radiation doses required for the observation of biological specimens in the electron microscope suggest t h a t the observation of unstained unshadowed macromolecules should be called microtephroscopy, the study of ashes with the microscope. In this light, our observation of fine-structure well below 10 A in the dark-field electron micrographs of myokinase and protamine becomes an exciting starting point from which to attempt the reconstruction of the biological structure of macromolecules at such a resolution. Moreover, the repeated observation of virtually identical images, as well as correlation with structure determined by X-ray crystallography argues that much of the detail observed is still biologically meaningful structure. INTRODUCTION
The examination of biological specimens in the electron microscope inevitably subjects them to enormous radiation doses. Isaacson et al. (4) have estimated that even with minimal irradiation techniques a dose of 100 electrons/A 2, or about 1011 rads, is required to realize a resolution of about 10 /k in the image. With such a dose one would assume that very little meaningful information at any level, not to speak of 10 Jr, is retained. Their measurements, using energy-loss spectra and electron diffraction, indicate that within a factor of 10 of such doses a large number of biological substances retain on the average about a third of their structural integrity (4). This result can be interpreted in several ways. Pessimistically, one can say most of the biological specimen is destroyed. Optimistically, however, some of the specimen is still intact. Moreover, if one supposes that loss of native electronic structure does not necessarily mean loss of all material, nor that loss of three-dimensional order means loss of two-dimensional projected detail, then an interesting possibility remains. Let the biological structure under the intense ra1Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
diation crumble into a heap of carbon, nitrogen, and oxygen atoms. In some way this heap of atomic ashes must bear a relationship to the specimen from which it was produced. The question that must be answered is how tenuous this relationship is, or to what resolution a biologically significant structural resemblance is maintained. In recognition of the fact that we are only perceiving the crumbling remains of the biological specimen, we have chosen to call this study microtephroscopy, a close look at the ashes. The results presented here indicate that with care, dark-field images of unstained and unshadowed proteins exhibit structural detail that is well below 10 A. Moreover, in one instance, x-ray crystallographic structure determination shows that much of this detail is biologically meaningful structure. Early work in dark field (5) had already indicated that helices in DNA, loops in tRNA, and the cleft in ribonuclease could be seen. It could be argued, however, that prior knowledge of such well-studied structures introduced a degree of subjectivity into the search for images resembling the desired configurations. Because the destruction wrought by the radiation dose mentioned above prevented the observa193
Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
194
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t i o n of large fields of w e l l - p r e s e r v e d i d e n t i cal s p e c i m e n s , s u c h a r g u m e n t s c o u l d n o t be c o u n t e r e d easily. S e v e r a l years ago to d i s p e l s u c h d o u b t s in our l a b o r a t o r y a n d to test our consistency in arriving at struct u r e s , we i n v e s t i g a t e d d a r k - f i e l d i m a g e s of m y o k i n a s e , a p r o t e i n whose s t r u c t u r e was unknown at that time. Subsequent x-ray a n a l y s i s a t low r e s o l u t i o n (6) r e a s s u r e d u s of t h e c o r r e c t n e s s of o u r gross s t r u c t u r a l a s s i g n m e n t . I n a d d i t i o n , however, reexa m i n a t i o n a n d c o m p a r i s o n of our i m a g e s w i t h r e c e n t h i g h r e s o l u t i o n x - r a y d a t a (7) often indicated a structural identity at levels b e t w e e n 6 a n d 10 /~. W e h a d origin a l l y d i s r e g a r d e d s u c h fine s t r u c t u r e as b e i n g m e a n i n g l e s s i n l i g h t of t h e r a d i a t i o n burden. T h e s e r e t r o s p e c t i v e d a t a on m y o k i n a s e are d e t a i l e d i n t h i s p a p e r . H o w e v e r , elect r o n m i c r o g r a p h s of a s e c o n d p r o t e i n , herr i n g s p e r m p r o t a m i n e , are s h o w n as well. T h e s e i m a g e s a g a i n show r e p r o d u c i b l e rep e a t e d d e t a i l a t a r e s o l u t i o n of 5-10 ~k. For this protein no independent confirmation of t h e s t r u c t u r e h a s b e e n c a r r i e d o u t as yet. METHODS AND MATERIALS Myokinase (pig muscle, Sigma Chemical Co., St. Louis, Mo.) was used at 0.1-10 #g/ml in 0.2 M ammonium acetate. Herring protamine sulphate (Mann Research Laboratories, New York) was separated into fractions Y-II, Y-I, and Z (3) and was desalted by loading on a CM-eellulose column in the protonated form, washing with distilled water, and eluting with 0.2 M HC1. The fractions were lyophilized and redissolved in distilled water for electron microscopy. Myokinase was examined on a Philips EM300 electron microscope nominally at 51 300X, protamine on a Siemens Elmiskop 102 at 50 000 and 100 000X. Magnifications were calibrated and corrected using a grating replica (Fullam, Schenectady, N. Y.). Both specimens were supported on thin carbon films ( < 20 ~) on fenestrated plastic supports (8). To obtain the minimum possible exposure a technique was followed that differed slightly for each of the two instruments. On the EM300 the beam at cross-over on the screen was carefully centered mechanically with the deflection system off. The deflection system was then reengaged and centered as well. With the beam still condensed to cover only the viewing screen so that no extraneous region would
receive an exposure, the microscope was now focused on an area of the specimen adjacent to the one to be photographed. The beam was electronically deflected to the edge of viewing screen so that only a mere sliver of the periphery of the screen was illuminated as an aid for the next step. The unirradiated specimen area was now moved into the center, a photo plate was transported under the screen, and the screen was lifted. Neither the desired specimen area nor the photographic plate were exposed at this point, since the beam was deflected off the field of view. After a few seconds, to permit mechanical vibrations to damp out, the exposure button was pressed while at the same time the electronic deflection system was shut off. This returned the beam to the mechanically aligned central position exposing the specimen and the plate. After the preset exposure time the shutter engaged and the plate was transported out of the way. On the Elmiskop 102 a second and independent control on the electronic deflection system was installed. Here the first control served to align the beam initially. The second control, which could be disengaged, then was used in a manner analogous to the deflection system of the EM300 above. RESULTS
M i n i m u m beam exposure. O u r t e c h n i q u e of m i n i m i z i n g e x p o s u r e to t h e specimen initially produced extremely frustrati n g r e s u l t s . S i n c e o n l y t h e a r e a a d j a c e n t to t h e one p h o t o g r a p h e d was u s e d to focus, we e x p e c t e d s o m e p l a t e s to be o u t of focus. A c h e c k o n t h e v i e w i n g s c r e e n after t h e i m a g e was r e c o r d e d r e v e a l e d t h a t a b o u t t h r e e o u t of four i m a g e s h a d b e e n f o c u s e d correctly. S u r p r i s i n g l y , however, a large n u m b e r of p l a t e s i n d i c a t e d m o v e m e n t or s t r e a k i n g of t h e i m a g e , s o m e t i m e s i n one d i r e c t i o n , t h e n at another angle on the next plate, s o m e t i m e s f a n - s h a p e d as t h o u g h e m a n a t ing f r o m s o m e p o i n t off the p l a t e . C a r e f u l e x a m i n a t i o n of " g o o d " p l a t e s i n d i c a t e d that even these exhibited streaking radially a w a y f r o m a s t i g m a t i c i n - f o c u s r e g i o n i n t h e c e n t r a l p o r t i o n of t h e b e a m . Fig. 1 shows a n e x a m p l e of t h i s p h e n o m e n o n . If a s e c o n d e x p o s u r e is m a d e of t h e i d e n t i c a l area, l e a v i n g t h e b e a m on t h e s p e c i m e n , the second micrograph appears perfectly n o r m a l , s h o w i n g n o n e of t h e effect. W e c a n o n l y s u g g e s t t h a t w h a t we o b s e r v e is t h e t r a n s i e n t t h a t occurs as t h e u n e x p o s e d cold u n c h a r g e d region heats up a n d charges
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FIo. 1. Typical appearance of radial streaking observed under our minimum exposure conditions in dark field. The center of the beam is revealed by the usefuI area of the micrograph slightly below and left of center. The specimen is myokinase on a thin carbon support, x 79 000. under the action of the beam. Once equilibrium is reached, the image remains steady. U n f o r t u n a t e l y by this time the specimen has received more t h a n a minimal dose, resulting in disappearance of fine detail in the structure, often beyond.a 20 A resolution, r e a r r a n g e m e n t of mass, and the a p p e a r a n c e of intense bright spots. Therefore, to preserve as m u c h fine structure as possible we have accepted the smaller useful field of view in the central portion of the b e a m and the need for careful centering of this b e a m on the camera of the microscope. Myokinase. T o obtain a measure of our consistency in determining unknown structures of macromolecules three of us two years ago i n d e p e n d e n t l y scanned five original plates of images of myokinase. These micrographs had been taken using different concentrations of the molecule and had been checked to assure that they were
in focus. The sole known facts were the molecular weight of the protein (22 000) and the magnification of the plates. Our aim was to ascertain independently the shape of the molecule, with about 25 to 30 examples each, m a r k e d on the plates to back up the structural assignment. All three of us arrived at a C-shaped molecule about 50 A in size, with one arm of the C slightly less dense than the other. Moreover, although we had looked at the same plates, none of us had chosen the same molecules. The structures picked generally were well isolated from one another, and, though of low contrast, were significantly different from the clean though motley carbon support film. An example of an area containing a p u r p o r t e d myokinase structure is shown in Fig. 2. This grayish white structure is distinct in shape, size, and contrast from background. However, only repeated observation of this type of struc-
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FIG. 2. Large area dark field image containing a single molecule of myokinase (arrow). The shape and contrast are distinctly different from background mottle. × 1 100 000.
ture as well as of the r a n d o m mottle of e m p t y carbon films can instil such confidence. This and other examples at higher magnification are shown in Fig. 3. These images are shown in reverse contrast since an intermediate enlargement was used to achieve this magnification. The C-shape generally predominates, though dumbbell structures (possibly a different projected view) as well as nondescript shapes are also found (bottom row, Fig. 3). We accepted a fairly noncontroversial image resolution of about 20 A at this stage and were pleased when low resolution x-ray diffraction studies suggested t h a t our assignment of the size and C-shape to myokinase was not only consistent b u t correct
(6). Recent x-ray studies at 3 A (7) made us
reexamine our work. M a n y of our images had a distinct loop or appendage on one side or the other near the b o t t o m (Fig. 3 ) . In addition, there was the occasional loop in the arms of the C-shape. One very striking example is enlarged in Fig. 4a alongside the drawing of the polypeptide chain of the molecule d e t e r m i n e d from x-ray analysis. If one considers t h a t the outside dimension of the molecule is 50 A then the observed structure in the image exhibits a resolution of 10 A or better. Moreover, since comparison with the x-ray d e t e r m i n a t i o n indicates a striking similarity, one is t e m p t e d to conclude t h a t the observed structure corresponds to biologically meaningful detail. P r o t a m i n e . T h e smallest proteins we have e x a m i n e d to date are the herring
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Fla. 3, Examples of shapes observed on dark field micrographs of myokinase. Reverse contrast, x 2 600 000.
@ 4b FIG. 4. Comparison of myokinase structure observed in dark field with the structure determined by X-ray crystallography [taken from (6) with permission]. × 4 200 000.
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sperm protamines. They have a molecular weight of about 4100 and have 20 or 21 arginine residues out of their 30 or 31 total amino acids. Their primary sequence is known but their structure has not yet been determined (1, 2). Our dark field micrographs indicate that the unstained unshadowed molecules are basically ellipsoidal in shape about 25 A in the shorter dimension and 35-40 A in the longer. Closer examination, however, reveals a substructure that is fan-shaped, comb-shaped, or star-shaped, often with six major leaves, tines, or rays. One such example is shown in the large area view in Fig. 5. It is possible that other prominent light areas in the figure may represent further molecules. However, these were too nondescript, possibly too
degraded, to merit further consideration, or their general shape and structure was not found repeatedly in other areas. Reproducibility and consistency in structure in a size range that could correspond to a molecular weight of 4100 were virtually the only criteria we could apply in this structure determination. For sizes larger or smaller than the above dimensions no consistent structures could be found at all. Within that size range, however, the task was relatively easy. Fig. 6a shows examples found in fraction Y-II, Fig. 6b molecules from fraction Y-I, and Fig. 6c from fraction Z. As for Figs. 3 and 4, these high magnification images are shown in reverse contrast. Although we have not made an exhaustive study, there appears to be little difference in structure between
FIG. 5. Large area dark field micrograph of a spread of protamine molecules. One image (marked by the arrow) is clearly characteristically different from random background. Other spots are rich enough in contrast to be distinct from background, but do not show enough detail to be useful for structure determination. × 1 100 000.
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FIG. 6. Examples of structurally interesting shapes observed in dark field micrographs of herring sperm protamine: (a) fraetion Y-II; (b) fraction Y-I; (c) fraction Z. Reverse contrast. × 3 500 000. the fractions. T h e similarity on the other h a n d is m u c h more striking. The structures are not all identical. Nevertheless, they can be easily related, assuming a small a m o u n t of flexibility in the molecule.
While here we c a n n o t yet claim fine s t r u c t u r e with biological significance, whatever has r e m a i n e d of the p r o t a m i n e molecule exhibits a reproducible structure with detail frequently at the 5 A level.
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Two examples, which are virtually identical in every detail, are shown in Fig. 7. For ease of m e a s u r e m e n t the scale here is 2 A/mm. These molecules came from different fractions on the gradient (though both are probably Y-I) and were taken at different electron optical magnifications (Fig. 7a at × 100 000, Fig. 7b at × 50 000). Since the radiation burden for the two images differed approximately by a factor of four, the similarity in structure is all the more remarkable. Nor are they isolated examples. The first image in Fig. 6 could almost be added to this pair, while several other structures in Fig. 6, though different from these, resemble each other quite closely. DISCUSSION We have e x a m i n e d dark field images of two proteins in an a t t e m p t to decide if meaningful structure d e t e r m i n a t i o n with the electron microscope at a resolution level of b e t t e r t h a n 20 A is possible. Our micrographs indicate t h a t reproducible detail is visible even at a 5 A level in m a n y images. Since comparison with x-ray d a t a has indicated for myokinase t h a t at least some of this detail represents biological structure, why should some of the detail in the images of p r o t a m i n e not also be biologically significant? If the electron b o m b a r d m e n t forms nonvolatile radicals or excited states in the macromolecule, the molecule can either do nothing, or react internally or with the carbon support. Gross rearrange-
ments of structure very likely would not be reproduced consistently. Such configurations would be discarded in the search for repeatedly similar structures. Minor alterations such as reactions with a neighboring amino acid side chain, even cross-linking with a close peptide chain in the same molecule would not necessarily be readily recognizable; nor would reactions with an adjacent portion of the carbon support film. Such a reaction might even stabilize the macromolecule a m o m e n t longer. As long as the two-dimensional projection of the mass of the molecule remains relatively constant, whatever the vertical rearrangem e n t might be, the electron micrograph would present a relatively faithful reproduction. Moreover, the repeated observation of virtually identical fine structure well below 10 A argues t h a t in these instances the structural alteration is probably minor, rather t h a n t h a t c o m m o n gross structural modifications have resulted in an identical end product. Our bias is obvious. We propose t h a t the finger-like segments of the images of protamine, often separated by as little as 5 A, are biologically significant. Proof of this on the one hand m u s t wait for x-ray crystallographic data. On the other hand, further electron microscopic studies of a modified protamine, the phosphorylated species, or a molecule m a r k e d with a specific localized cluster of heavy atoms m a y also shed some light on the verity of our proposal. T h e latter approach has already been shown to
FIG. 7. Two dark field micrographs of protamine that are virtually identical in every detail showing structure with measurable 5/~ image resolution: (a) from fraction Y-I, taken at × 100 000; (b) from fraction Y-I, taken at × 50 000. Since the final magnification is the same, the difference in electron optical magnification accounts for the difference in granularity of the images. × 5 000 000.
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be feasible in the case of transfer RNA (Korn and Ottensmeyer, in preparation). Finally, however, we think that our results have shown that too pessimistic an approach to the resolution problem is not warranted. A careful examination of the images of even crumbling biological structures may reveal much about their original high resolution detail. This work was supported by the National Cancer Institute of Canada and grant MA3763 of the National Research Council of Canada. The second author held an NCI of Canada Fellowship. Figure 4b was kindly provided by Drs. G. E. Schulz and R. H. Schirmer.
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REFERENCES 1. ANDO,T., ANDSUZUKI,K., Biochim. Biophys. Acta 121, 427 (1966). 2. ANDO,T., ANDSUZUKI,K., Biochim. Biophys. Acta 140, 375 (1967). 3. ANDO, T., AND WATANABE,S., Internat. J. Protein Res. l, 221 (1969). 4. ISAACSON, M., JOHNSON, D., AND CREWE, A. V., Radiat. Res. 55, 205 (1973). 5. OTTENSMEYER,f. P., Biophys. J. 9, 1144 (1969). 6. SCHULZ,G. E., BIEDERMANN,K., KABSCH,W., AND SCHIRMER, R. H., J. Mol. Biol. 80, 857 (1973). 7. SCHULZ,G. E., ELZINGA,M., MARX, F., ANDSCHIRMER, R. H., Nature 250, 120 (1974). 8. WHITING, R. F., AND OTTENSMEYER,F. P., J. Mol. Biol. 67, 173 (1972).