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Structure and dimensions of bacteriophage φX174 from electron microscopy
J. Mol . Biol., (1959), 1, 192-194
Structure and Dimensions of Bacteriophage ¢X174 from Electron Microscopy We have prepared samples of bacterioph age .pX174 (Sinsheimer, 1959; Tessman, 1959) for electron microscopy by the following pr ocedure. The phage, obtained originally from Dr. R. L. Sinsheimer, was grown on Escherichia coli st ra in 0 , in synthetic medium buffered with tris-HOI at pH 7·4. Th e lysed cult ure was centrifuged at low speed t o remov e cell debris. After making the supernatant 3 !ll in ammonium sulfate , the phage was precipitated by centrifuging at 15,000 rev/min. The pellet was resuspended in 0·01 M-ammonium acetate, pH 7,4, and adsorbed on a diethylaminoethylcellulose anion exchange column. The phage was eluted from the column by 0·10 M-ammonium acetate. The purification of larger quantities of viru s ha s subsequ ently been carri ed out by modifications of the above procedure. Although .pX174 is large enough t o be readily visible by the techniques of conven tional electron micro scopy, our specimens were prepared by a method developed for the observation of much smaller particles. Th e phage were support ed on the surface of freshly cleaved mica, and sha dowcast with platinum which was lat er stripped from the mica (Hall, 1956). A typical micrograph is shown in Plate I (a) where several characteristi c features are evident: (i) ea ch virus has a high spot in the center ; (ii) the shadows ar e polygonal and sometimes a sharp spike may be discerned corresponding to the high central point; (iii) the periphery of the virus is seen to consist of granules or "knobs" arranged as a t the corners of a polyhedron, but the shadowing angl e (shadow-to.height ratio 2·75 : 1) is such that the fine st ru ct ure is obscured on the side fa cing the evaporating source . In Pl ate I (b), at high er magnification and at a shadow-to -height ratio of ab out 1 : 1, the fine struc t ure is better resolved although there is the illusion that the particles are flattened. H ere one sees that the central high point is surrounded by five granules arran ged with pentagonal sym metry. On the side of t he virus fa cing the evaporation sourc e a lower tier of granules also intercepts metal which tends to fill in the space between the members of the upper five. The presence of a second lower ti er may also be inferred from the fact that the height of the upper tier, as deduced fr om shadow lengths, is ab out equal to the distance along the two granules forming the side of the pentagonal pattern. Except for a few particles which appear to hav e disin te grated and those that are in cont act with each ot her , all particles pr esent the same aspect, when properly shadowed, as illustrated in Plate I (b). We have seen no evid ence for tails, attached or det ached. A micrograph of a specimen prepared by a negative st aining t echnique (Horne & Brenner, 1958; Hall, 1955) is illustrated in Plate I (c). In such micrographs the virus outlines are polyhedral. We have also made micro graphs of a purified sample of .pX174 obtained directly from Dr. R. L. Sinsheimer and have observed the same morphological features described above both by shadow casting and negative staining techniques. Measurements of particle diameters (assuming a roughly spherical shape) from 192
1°21'
PLATE I (a). B a cterio ph age .pX1 74 on a m ica su bs t ra te shadowed wi th pl a tinum a t a shadowto -height ratio of about 3 : l. :Magn ifica t ion 106, 000 X . (b) .pX 174 sh adowed a t an angle I : I sh own a t va rious a zimut hs with resp ect to the direction of sha dowing. Maghificat.ion 190,000 X . (c) .pX174 dried down in n eutral phosphotungstic ac id . Magnification 164,000 X.
[ToJace page 192.
LETTERS TO THE EDITOR
193
shadowed specimens may be made in two ways: (a) measurement of apparent particle diameter perpendicular to the direction of shadowing, including the cap of metal, and (b) measurement of shadow lengths and calculation of particle heights from shadow-toheight ratios obtained from polystyrene spheres in the same field. Results of an unpublished study by one of us (C.E.H.) indicate that under the standardized conditions used the apparent diameters of approximately spherical particles including the cap of metal are too large by 50 to 75 A, while the shadow lengths are a good measure of particle diameter. Measured diameters of shadowed cj>X174 are about 50 A greater than heights which would be expected for a spherical particle, indicating that there is negligible flattening due to drying forces. The average of all determinations from shadow lengths and from diameters minus 50 A for the metal, of the M.LT. virus and that obtained from Dr. Sinsheimer, is 248 A with a mean deviation of ± 10 A. Averages for the diameters from the two sources are 243 A and 252 A respectively which are within experimental error of the overall mean. Diameters from negatively stained preparations are slightly smaller than from shadowed preparations but in these micrographs there is some difficulty in locating the particle edges accurately. If a sphere were to contain the amount of protein and deoxyribonucleic acid (DNA) that has been determined for cj>X174 by Sinsheimer (1959) (4,6 X 10 6 mol wt units of protein of density 1·3 and 1·7 X 10 6 mol wt units of DNA of density 1,7) the sphere would have a diameter of 240 A, in good agreement with the electron microscope results. The regular structural pattern of the shadowed phage, as well as the polygonal outline of the shadows and of the negatively stained particles, strongly suggests that the structure of the phage is related to a regular polyhedron. Williams & Smith (1958) have demonstrated that T'ipula Iridescent virus, of approximately 1300 A diameter, has the shape of an icosahedron with faces that are planar rather than granular as with cj>X174. Angularities in outline of small viruses have also been noted by Williams (1954) and Kaesberg (1956), and Sinsheimer (1959) has noted occasional evidence for a polyhedral shape in micrographs of cj>X174. A likely interpretation of our pictures of cj>X174 is that the phage contains a shell of twelve "knobs" arranged regularly as on the faces of a regular dodecahedron. This is based on the observation of one apical and five surrounding peripheral knobs, with the suggestion of a second underlying tier similarly arranged. This interpretation implies that every phage in the shadowed pictures would have to rest on the knob opposite the central apex seen in every particle. It is possible that upon drying there would be some distortion of the phage that would provide a broader base of contact. The alternative explanation that the phage is not regular but contains a broad face appears unlikely because such a particle would be expected to present a variety of appearances in shadow-cast micrographs, unless the broad face preferentially adsorbs to the substrate. If cj>X174 does contain twelve regularly arranged knobs one might expect that the phage could sometimes orient itself with three knobs in contact with the substrate. This would produce both a narrower shadow near the base and a pattern of highlights which we do not observe. The outline of negatively-stained virus is consistent with the outlines of the type of polyhedron postulated although the resolution is not adequate to establish the shape unequivocally. Crick & Watson (1956) have speculated that many small viruses may be made up of subunits, related by symmetry elements. The electron microscope pictures give suggestive evidence that this is true for cj>X174. In particular, twelve regularly
194
C . E. HALL , E . C . MACLEAN AND 1. TE SS M A N
arran ged knobs would be relat ed at least by four three-fold and three two-fold rotation axes of symmetry. The pi ctures are consiste nt with the existe nce of five-fold symmetry ax es, as well, but thi s would require that each knob contained a multiple of five symmetrically arranged su bunits, about which we have no evidence . We believe these pictures confirm the suggest ion of common structural features of small viruses, whether plant, animal or bacterial. This inv estigation was sup p ort ed by r esearch gr ant C-2171 (C6 and 7) fr om the National Cancer I nstitute, by training gran t 2A-l (P .B .) (C) fr om the N ational Institute of Arthriti s and Met abolic Di seases, N ation al I nst it utes of H ealth, United St ates Public H ealth Ser vic e, and by re search grant E-2128 (Cl) from the N ation al Institute of Allergy an d I n fectious Di seases, United States Public Health Service. C EC IL
E.
HALL
ELIZABETH C. MACLEAN IRWIN TESSMAN
D epartment of Biology Massachuset t a Institut e of Te chnology Cam b rid ge, Mass., U.S.A. R eceived 23 February 1959
REFERENCES Cri ck, F . H . C. & Watson, J. D . (1956). Nature, 177, 473 . H all, C. E . (1955). J. Biophys. B iochem. Oytol. 1, 1. H all, C. E . (1956). J. Biophys . B iochem. Oytol. 2, 625. Horne, R. W . & Brenner, S. (1958). Programme of 4th International Conference on El ectron Microscopy, p. 212. Berl in . K a esb er g, P . (1956). Science, 124, 626. Sinsheimer, R. L . (1959). J . Mol. B iol. 1, 37. T essman, 1. (1959). Virology, 7, 263. Williams, R. C. (1954). Admnc. Virus Res . 2, 183. Williams, R. C. & Smith, K. M. (1958). Biochim biophys. Acta, 28, 469.
Structure and Dimensions of Bacteriophage ¢X174 from Electron Microscopy We have prepared samples of bacterioph age .pX174 (Sinsheimer, 1959; Tessman, 1959) for electron microscopy by the following pr ocedure. The phage, obtained originally from Dr. R. L. Sinsheimer, was grown on Escherichia coli st ra in 0 , in synthetic medium buffered with tris-HOI at pH 7·4. Th e lysed cult ure was centrifuged at low speed t o remov e cell debris. After making the supernatant 3 !ll in ammonium sulfate , the phage was precipitated by centrifuging at 15,000 rev/min. The pellet was resuspended in 0·01 M-ammonium acetate, pH 7,4, and adsorbed on a diethylaminoethylcellulose anion exchange column. The phage was eluted from the column by 0·10 M-ammonium acetate. The purification of larger quantities of viru s ha s subsequ ently been carri ed out by modifications of the above procedure. Although .pX174 is large enough t o be readily visible by the techniques of conven tional electron micro scopy, our specimens were prepared by a method developed for the observation of much smaller particles. Th e phage were support ed on the surface of freshly cleaved mica, and sha dowcast with platinum which was lat er stripped from the mica (Hall, 1956). A typical micrograph is shown in Plate I (a) where several characteristi c features are evident: (i) ea ch virus has a high spot in the center ; (ii) the shadows ar e polygonal and sometimes a sharp spike may be discerned corresponding to the high central point; (iii) the periphery of the virus is seen to consist of granules or "knobs" arranged as a t the corners of a polyhedron, but the shadowing angl e (shadow-to.height ratio 2·75 : 1) is such that the fine st ru ct ure is obscured on the side fa cing the evaporating source . In Pl ate I (b), at high er magnification and at a shadow-to -height ratio of ab out 1 : 1, the fine struc t ure is better resolved although there is the illusion that the particles are flattened. H ere one sees that the central high point is surrounded by five granules arran ged with pentagonal sym metry. On the side of t he virus fa cing the evaporation sourc e a lower tier of granules also intercepts metal which tends to fill in the space between the members of the upper five. The presence of a second lower ti er may also be inferred from the fact that the height of the upper tier, as deduced fr om shadow lengths, is ab out equal to the distance along the two granules forming the side of the pentagonal pattern. Except for a few particles which appear to hav e disin te grated and those that are in cont act with each ot her , all particles pr esent the same aspect, when properly shadowed, as illustrated in Plate I (b). We have seen no evid ence for tails, attached or det ached. A micrograph of a specimen prepared by a negative st aining t echnique (Horne & Brenner, 1958; Hall, 1955) is illustrated in Plate I (c). In such micrographs the virus outlines are polyhedral. We have also made micro graphs of a purified sample of .pX174 obtained directly from Dr. R. L. Sinsheimer and have observed the same morphological features described above both by shadow casting and negative staining techniques. Measurements of particle diameters (assuming a roughly spherical shape) from 192
1°21'
PLATE I (a). B a cterio ph age .pX1 74 on a m ica su bs t ra te shadowed wi th pl a tinum a t a shadowto -height ratio of about 3 : l. :Magn ifica t ion 106, 000 X . (b) .pX 174 sh adowed a t an angle I : I sh own a t va rious a zimut hs with resp ect to the direction of sha dowing. Maghificat.ion 190,000 X . (c) .pX174 dried down in n eutral phosphotungstic ac id . Magnification 164,000 X.
[ToJace page 192.
LETTERS TO THE EDITOR
193
shadowed specimens may be made in two ways: (a) measurement of apparent particle diameter perpendicular to the direction of shadowing, including the cap of metal, and (b) measurement of shadow lengths and calculation of particle heights from shadow-toheight ratios obtained from polystyrene spheres in the same field. Results of an unpublished study by one of us (C.E.H.) indicate that under the standardized conditions used the apparent diameters of approximately spherical particles including the cap of metal are too large by 50 to 75 A, while the shadow lengths are a good measure of particle diameter. Measured diameters of shadowed cj>X174 are about 50 A greater than heights which would be expected for a spherical particle, indicating that there is negligible flattening due to drying forces. The average of all determinations from shadow lengths and from diameters minus 50 A for the metal, of the M.LT. virus and that obtained from Dr. Sinsheimer, is 248 A with a mean deviation of ± 10 A. Averages for the diameters from the two sources are 243 A and 252 A respectively which are within experimental error of the overall mean. Diameters from negatively stained preparations are slightly smaller than from shadowed preparations but in these micrographs there is some difficulty in locating the particle edges accurately. If a sphere were to contain the amount of protein and deoxyribonucleic acid (DNA) that has been determined for cj>X174 by Sinsheimer (1959) (4,6 X 10 6 mol wt units of protein of density 1·3 and 1·7 X 10 6 mol wt units of DNA of density 1,7) the sphere would have a diameter of 240 A, in good agreement with the electron microscope results. The regular structural pattern of the shadowed phage, as well as the polygonal outline of the shadows and of the negatively stained particles, strongly suggests that the structure of the phage is related to a regular polyhedron. Williams & Smith (1958) have demonstrated that T'ipula Iridescent virus, of approximately 1300 A diameter, has the shape of an icosahedron with faces that are planar rather than granular as with cj>X174. Angularities in outline of small viruses have also been noted by Williams (1954) and Kaesberg (1956), and Sinsheimer (1959) has noted occasional evidence for a polyhedral shape in micrographs of cj>X174. A likely interpretation of our pictures of cj>X174 is that the phage contains a shell of twelve "knobs" arranged regularly as on the faces of a regular dodecahedron. This is based on the observation of one apical and five surrounding peripheral knobs, with the suggestion of a second underlying tier similarly arranged. This interpretation implies that every phage in the shadowed pictures would have to rest on the knob opposite the central apex seen in every particle. It is possible that upon drying there would be some distortion of the phage that would provide a broader base of contact. The alternative explanation that the phage is not regular but contains a broad face appears unlikely because such a particle would be expected to present a variety of appearances in shadow-cast micrographs, unless the broad face preferentially adsorbs to the substrate. If cj>X174 does contain twelve regularly arranged knobs one might expect that the phage could sometimes orient itself with three knobs in contact with the substrate. This would produce both a narrower shadow near the base and a pattern of highlights which we do not observe. The outline of negatively-stained virus is consistent with the outlines of the type of polyhedron postulated although the resolution is not adequate to establish the shape unequivocally. Crick & Watson (1956) have speculated that many small viruses may be made up of subunits, related by symmetry elements. The electron microscope pictures give suggestive evidence that this is true for cj>X174. In particular, twelve regularly
194
C . E. HALL , E . C . MACLEAN AND 1. TE SS M A N
arran ged knobs would be relat ed at least by four three-fold and three two-fold rotation axes of symmetry. The pi ctures are consiste nt with the existe nce of five-fold symmetry ax es, as well, but thi s would require that each knob contained a multiple of five symmetrically arranged su bunits, about which we have no evidence . We believe these pictures confirm the suggest ion of common structural features of small viruses, whether plant, animal or bacterial. This inv estigation was sup p ort ed by r esearch gr ant C-2171 (C6 and 7) fr om the National Cancer I nstitute, by training gran t 2A-l (P .B .) (C) fr om the N ational Institute of Arthriti s and Met abolic Di seases, N ation al I nst it utes of H ealth, United St ates Public H ealth Ser vic e, and by re search grant E-2128 (Cl) from the N ation al Institute of Allergy an d I n fectious Di seases, United States Public Health Service. C EC IL
E.
HALL
ELIZABETH C. MACLEAN IRWIN TESSMAN
D epartment of Biology Massachuset t a Institut e of Te chnology Cam b rid ge, Mass., U.S.A. R eceived 23 February 1959
REFERENCES Cri ck, F . H . C. & Watson, J. D . (1956). Nature, 177, 473 . H all, C. E . (1955). J. Biophys. B iochem. Oytol. 1, 1. H all, C. E . (1956). J. Biophys . B iochem. Oytol. 2, 625. Horne, R. W . & Brenner, S. (1958). Programme of 4th International Conference on El ectron Microscopy, p. 212. Berl in . K a esb er g, P . (1956). Science, 124, 626. Sinsheimer, R. L . (1959). J . Mol. B iol. 1, 37. T essman, 1. (1959). Virology, 7, 263. Williams, R. C. (1954). Admnc. Virus Res . 2, 183. Williams, R. C. & Smith, K. M. (1958). Biochim biophys. Acta, 28, 469.