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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas a
b
Julyet Aksiyote-Benbasat , George C. Ruben & Kenneth A. Marx
c
a
Department of Microbiology , University of British Columbia , #300-6174 University Blvd., Vancouver , B.C. , Canada , V6T 1W5 b
Department of Biological Sciences , Dartmouth College , Hanover , N.H. , 03755 c
Department of Chemistry , University of Lowell , One University Ave., Lowell , MA , 01854 Published online: 21 May 2012.
To cite this article: Julyet Aksiyote-Benbasat , George C. Ruben & Kenneth A. Marx (1990) Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas, Journal of Biomolecular Structure and Dynamics, 7:4, 773-794, DOI: 10.1080/07391102.1990.10508523 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508523
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the
Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [New York University] at 11:00 16 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 7, Issue Number 4 (1990), ®Adenine Press (1990).
Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas Julyet Aksiyote-Benbasat 1, George C. Ruben 2 and Kenneth A. Marx? Downloaded by [New York University] at 11:00 16 May 2015
1
Department of Microbiology University of British Columbia #300-6174 University Blvd. Vancouver, B.C., Canada V6T 1W5 2
Department of Biological Sciences Dartmouth College Hanover, N.H. 03755 3
Department of Chemistry University of Lowell One University Ave. Lowell, MA 01854
Abstract The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80° angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q numberof21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with . 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.
Introduction Bacteriophage T4 is one of the most complicated of the tailed bacteriophage. It has
773
Downloaded by [New York University] at 11:00 16 May 2015
774
Aksiyote·Benbasat eta/.
been extensively studied by a combination of genetic, molecular biological, biochemical and structural techniques. We are concerned with phage structure, and a number of reviews of this field have been published recently (1-3). The principal features of the icosahedral capsid geometry of all T -even bacteriophage have been established by a combination of analytical biochemistry, electron microscopy and symmetry considerations. Until recently the most persuasive demonstration of the icosahedral capsid geometry was achieved by counting capsomeres in bacteriophage T2 under low angle shadowing conditions (4). Because of the presence of two additional proteins, hoc and soc, on the T4 head, capsomers have never been visualized on T4 (5). More recently, while this work was in progress, a few of the icosahedral triangular faces were visualized in T2 (5). In the present study we demonstrate, for the first time, the direct visualization of all equilateral triangles in both pentameric caps as well as the elongated isosceles triangular faces that form the cylindrical part of the prolate icosahedron. By making measurements on individual deep-etched T4, such as the head length to head width, the angle through which the pentameric head and tail pentameric caps are rotated with respect to each other to become perfectly aligned and the isoseles triangular face angle, we have estimated a Q number for the T4 capsid size by also using the generally accepted triangulation number, T, of 13 (4,5). There have been numerous studies in the past decades in which bacteriophage T4 and subassemblies of T4 have been studied by transmission electron microscopy (TEM) (1). These studies have all been largely based on the negative staining preparation technique forTEM. The large number of published studies have fostered a consensus view of the solution conformation of bacteriophage T4. In this study we demonstrate that a number of T4 structural features seen in negative staining studies are different in our freeze-fractured/ deep-etched (FD ET)/vertically platinumcarbon (Pt-C) replicated phage preparations. At least one of the FDET observed features more accurately reflects the solution conformation ofT4 phage. The reason for this is that our protocol allows the particles to be viewed from any perspective as they occur supended in the ice, whereas negatively stained particles always lie on their long sides in contact with the grid surface. In addition, negative staining shows the best contrast where stain penetrates and around the edges of an object where the stain is thickest, while FDET/vertical replication yields more subtle contrast over the whole surface of the object. The new FDET/vertical Pt-C replication protocol used here has been shown to have at least 1.2 nm resolution with the capability of0.7 nm resolution (6). Vertical Pt-C deposition (80° angle) with 1 nm Pt-C films accurately defines the topology of all exposed surfaces of an object. The slope of each surface determines the final deposited metal film thickness which determines contrast. For instance the Pt-C film is thickest on horizontal surfaces and becomes progressively thinner as a surface slope approaches a vertical incline. This surface slope dependent Pt-C film thickness is how shape can be perceived in the images not viewed stereoscopically. We have generally viewed phage images in stereo to confirm the interpretations presented in single images. Stereoviewing generally resolves any ambiguous image interpretations that arise. Vertical Pt-C replication, unlike negative stains which surround and/or penetrate phage structure on a support film, only coats the exposed
Figure 1: Top view ofthe deep-etched wild type bacteriophage T40 capsid. This tracing was made from the right-hand image of the stereopair. The stereoimages were viewed so that all lines and vertices in the drawing were correctly identified showing a cap composed of five equilateral triangles. The tilt angle between stereoimages is 10°. The particle stippled on the drawing's five fold symmetry axis which is seen in the stereopair, might be the gp24 protein thought to be located at this position (7). Magnification 490,000X.
Downloaded by [New York University] at 11:00 16 May 2015
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Figure 2: Tracing ofthe deep-etched osmotic shock resistant bacteriophage T40, side view, was made from the left-hand image of the stereopair. The stereopair was viewed simultaneously so that all lines and vertices in the drawing were correctly identified. The tilt angle between the stereoimages is 10°. The drawing shows a series of adjacent isosceles triangles with first one triangle with its basal side bordering one pentameric cap followed by the next triangle with its basal side bordering the opposite pentameric cap. Magnification 500,000X.
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas a
b
Julyet Aksiyote-Benbasat , George C. Ruben & Kenneth A. Marx
c
a
Department of Microbiology , University of British Columbia , #300-6174 University Blvd., Vancouver , B.C. , Canada , V6T 1W5 b
Department of Biological Sciences , Dartmouth College , Hanover , N.H. , 03755 c
Department of Chemistry , University of Lowell , One University Ave., Lowell , MA , 01854 Published online: 21 May 2012.
To cite this article: Julyet Aksiyote-Benbasat , George C. Ruben & Kenneth A. Marx (1990) Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas, Journal of Biomolecular Structure and Dynamics, 7:4, 773-794, DOI: 10.1080/07391102.1990.10508523 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508523
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the
Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [New York University] at 11:00 16 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 7, Issue Number 4 (1990), ®Adenine Press (1990).
Solution Structure of Bacteriophage T4D and Icosahedral Capsid Geometry Visualized in Freeze-Fractured, Deep-Etched Replicas Julyet Aksiyote-Benbasat 1, George C. Ruben 2 and Kenneth A. Marx? Downloaded by [New York University] at 11:00 16 May 2015
1
Department of Microbiology University of British Columbia #300-6174 University Blvd. Vancouver, B.C., Canada V6T 1W5 2
Department of Biological Sciences Dartmouth College Hanover, N.H. 03755 3
Department of Chemistry University of Lowell One University Ave. Lowell, MA 01854
Abstract The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80° angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q numberof21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with . 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.
Introduction Bacteriophage T4 is one of the most complicated of the tailed bacteriophage. It has
773
Downloaded by [New York University] at 11:00 16 May 2015
774
Aksiyote·Benbasat eta/.
been extensively studied by a combination of genetic, molecular biological, biochemical and structural techniques. We are concerned with phage structure, and a number of reviews of this field have been published recently (1-3). The principal features of the icosahedral capsid geometry of all T -even bacteriophage have been established by a combination of analytical biochemistry, electron microscopy and symmetry considerations. Until recently the most persuasive demonstration of the icosahedral capsid geometry was achieved by counting capsomeres in bacteriophage T2 under low angle shadowing conditions (4). Because of the presence of two additional proteins, hoc and soc, on the T4 head, capsomers have never been visualized on T4 (5). More recently, while this work was in progress, a few of the icosahedral triangular faces were visualized in T2 (5). In the present study we demonstrate, for the first time, the direct visualization of all equilateral triangles in both pentameric caps as well as the elongated isosceles triangular faces that form the cylindrical part of the prolate icosahedron. By making measurements on individual deep-etched T4, such as the head length to head width, the angle through which the pentameric head and tail pentameric caps are rotated with respect to each other to become perfectly aligned and the isoseles triangular face angle, we have estimated a Q number for the T4 capsid size by also using the generally accepted triangulation number, T, of 13 (4,5). There have been numerous studies in the past decades in which bacteriophage T4 and subassemblies of T4 have been studied by transmission electron microscopy (TEM) (1). These studies have all been largely based on the negative staining preparation technique forTEM. The large number of published studies have fostered a consensus view of the solution conformation of bacteriophage T4. In this study we demonstrate that a number of T4 structural features seen in negative staining studies are different in our freeze-fractured/ deep-etched (FD ET)/vertically platinumcarbon (Pt-C) replicated phage preparations. At least one of the FDET observed features more accurately reflects the solution conformation ofT4 phage. The reason for this is that our protocol allows the particles to be viewed from any perspective as they occur supended in the ice, whereas negatively stained particles always lie on their long sides in contact with the grid surface. In addition, negative staining shows the best contrast where stain penetrates and around the edges of an object where the stain is thickest, while FDET/vertical replication yields more subtle contrast over the whole surface of the object. The new FDET/vertical Pt-C replication protocol used here has been shown to have at least 1.2 nm resolution with the capability of0.7 nm resolution (6). Vertical Pt-C deposition (80° angle) with 1 nm Pt-C films accurately defines the topology of all exposed surfaces of an object. The slope of each surface determines the final deposited metal film thickness which determines contrast. For instance the Pt-C film is thickest on horizontal surfaces and becomes progressively thinner as a surface slope approaches a vertical incline. This surface slope dependent Pt-C film thickness is how shape can be perceived in the images not viewed stereoscopically. We have generally viewed phage images in stereo to confirm the interpretations presented in single images. Stereoviewing generally resolves any ambiguous image interpretations that arise. Vertical Pt-C replication, unlike negative stains which surround and/or penetrate phage structure on a support film, only coats the exposed
Figure 1: Top view ofthe deep-etched wild type bacteriophage T40 capsid. This tracing was made from the right-hand image of the stereopair. The stereoimages were viewed so that all lines and vertices in the drawing were correctly identified showing a cap composed of five equilateral triangles. The tilt angle between stereoimages is 10°. The particle stippled on the drawing's five fold symmetry axis which is seen in the stereopair, might be the gp24 protein thought to be located at this position (7). Magnification 490,000X.
Downloaded by [New York University] at 11:00 16 May 2015
(')
CJ1
...... ......
~
c:
Cl)
~ :;c: (')
cg
.g i
~
.
~
i
Figure 2: Tracing ofthe deep-etched osmotic shock resistant bacteriophage T40, side view, was made from the left-hand image of the stereopair. The stereopair was viewed simultaneously so that all lines and vertices in the drawing were correctly identified. The tilt angle between the stereoimages is 10°. The drawing shows a series of adjacent isosceles triangles with first one triangle with its basal side bordering one pentameric cap followed by the next triangle with its basal side bordering the opposite pentameric cap. Magnification 500,000X.
Downloaded by [New York University] at 11:00 16 May 2015
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