The development of low-dose electron cryo-microscopy has provided the means to see structural det... more The development of low-dose electron cryo-microscopy has provided the means to see structural details to better than 10 A resolution in helical structures. The application of techniques of image analysis to micrographs can yield accurate phases, but not amplitudes with which to generate three-dimensional maps of the structure. Electron diffraction can provide reliable amplitudes, which can be combined with the phases from the images. In order to collect amplitude data, two problems have to be overcome: the pattern should be obtained from a large well ordered sample of particles, and the inelastic background should be properly subtracted. In this paper, we present three simple methods to produce rafts of helical particles. Using these methods we have obtained electron diffraction patterns from TMV (with data out to 0.28 nm), TMV protein stacked disks (with data out to 0.3 nm) and bacterial flagellar filaments (with data out to 0.5 nm). In addition, we describe the algorithms used to extract the amplitudes from the diffraction patterns.
Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 1970
Downloaded from rspa.royalsocietypublishing.org on November 5, 2010 320 RA Crowther, D. J. DeRosi... more Downloaded from rspa.royalsocietypublishing.org on November 5, 2010 320 RA Crowther, D. J. DeRosier and A. Klug orientations or by examining a single particle using a tilting stage or by a combina-tion of these methods. If sufficient data are available, the object can then ...
The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what w... more The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what we refer to as a "staircase-like" profile. At the proximal end of the cochlea the length of the tallest row of stereocilia in the staircase is 1.5 micron, with the shortest row only 0.3 micron. As one proceeds towards the distal end of the cochlea the length of the stereocilia progressively increases so that at the extreme distal end the length of the tallest row of the staircase is 5.5 micron and the shortest row is 2 micron. During development hair cells form their staircases in four phases of growth separated from each other by developmental time. First, stereocilia sprout from the apical surfaces of the hair cells (8-10-d embryos). Second (10-12-d embryos), what will be the longest row of the staircase begins to elongate. As the embryo gets older successive rows of stereocilia initiate elongation. Thus the staircase is set up by the sequential initiation of elongation of stereociliary rows located at increased distances from the row that began elongation. Third (12-17-d embryos), all the stereocilia in the newly formed staircase elongate until those located on the first step of the staircase have reached the prescribed length. In the final phase (17-d embryos to hatchlings) there is a progressive cessation of elongation beginning with the shortest step and followed by taller and taller rows with the tallest step stopping last. Thus, to obtain a pattern of stereocilia in rows of increasing height what transpires are progressive go signals followed by a period when all the stereocilia grow and ending with progressive stop signals. We discuss how such a sequence could be controlled.
An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some... more An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some filaments are surrounded by a set of three windings that form a sheath. We provide evidence that the sheath is composed of subunits having a molecular mass of 24,000 Da. We suggest that the sheath could be composed of protofilaments of flagellin wound around the filament.
Using a new procedure that combines electron-density correlation with biochemical information, we... more Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.
The size of the putative export channel in the bacterial flagellar filament appears small (25 A) ... more The size of the putative export channel in the bacterial flagellar filament appears small (25 A) in studies done by electron microscopy but large (60 A) in studies done by X-ray diffraction. We have undertaken additional studies by electron microscopy to examine some of the possible causes of the difference. A comparison of three-dimensional image reconstructions of native and reconstituted filaments rules out the presence or absence of flagellin monomers in the export channel as the source of the variation in apparent channel size. The channel seen in reconstructions from both kinds of filaments is 25 A in diameter. The difference in the previous studies is more probably a result of artifacts introduced in either the X-ray or the electron microscopical methodology. Comparisons of three-dimensional reconstructions from images of filaments embedded in various stains (anionic, cationic and neutral) and in ice, taken at a range of defocuses, rule out the two most likely sources of artifact in electron microscopy (i.e., staining artifacts and defocus phase contrast). Based on these studies we suggest that the channel seen in the image reconstructions is free of exported flagellin monomers, that its true diameter is about 25 A, and, therefore, that the flagellin monomer must be unfolded to pass along it.
Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signalin... more Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin-fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin-fimbrin bundles. Furthermore, the assembly of the actin-fimbrin arrays suggests coupling between actin polymerization, fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity.
Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are appr... more Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822-834) on the packing of actin filaments in this stereocilia.
Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin fil... more Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.
We have determined the structure of the actin-scruin filament to 13-A resolution using a combinat... more We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)-ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross-links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.
The development of low-dose electron cryo-microscopy has provided the means to see structural det... more The development of low-dose electron cryo-microscopy has provided the means to see structural details to better than 10 A resolution in helical structures. The application of techniques of image analysis to micrographs can yield accurate phases, but not amplitudes with which to generate three-dimensional maps of the structure. Electron diffraction can provide reliable amplitudes, which can be combined with the phases from the images. In order to collect amplitude data, two problems have to be overcome: the pattern should be obtained from a large well ordered sample of particles, and the inelastic background should be properly subtracted. In this paper, we present three simple methods to produce rafts of helical particles. Using these methods we have obtained electron diffraction patterns from TMV (with data out to 0.28 nm), TMV protein stacked disks (with data out to 0.3 nm) and bacterial flagellar filaments (with data out to 0.5 nm). In addition, we describe the algorithms used to extract the amplitudes from the diffraction patterns.
Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences, 1970
Downloaded from rspa.royalsocietypublishing.org on November 5, 2010 320 RA Crowther, D. J. DeRosi... more Downloaded from rspa.royalsocietypublishing.org on November 5, 2010 320 RA Crowther, D. J. DeRosier and A. Klug orientations or by examining a single particle using a tilting stage or by a combina-tion of these methods. If sufficient data are available, the object can then ...
The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what w... more The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what we refer to as a "staircase-like" profile. At the proximal end of the cochlea the length of the tallest row of stereocilia in the staircase is 1.5 micron, with the shortest row only 0.3 micron. As one proceeds towards the distal end of the cochlea the length of the stereocilia progressively increases so that at the extreme distal end the length of the tallest row of the staircase is 5.5 micron and the shortest row is 2 micron. During development hair cells form their staircases in four phases of growth separated from each other by developmental time. First, stereocilia sprout from the apical surfaces of the hair cells (8-10-d embryos). Second (10-12-d embryos), what will be the longest row of the staircase begins to elongate. As the embryo gets older successive rows of stereocilia initiate elongation. Thus the staircase is set up by the sequential initiation of elongation of stereociliary rows located at increased distances from the row that began elongation. Third (12-17-d embryos), all the stereocilia in the newly formed staircase elongate until those located on the first step of the staircase have reached the prescribed length. In the final phase (17-d embryos to hatchlings) there is a progressive cessation of elongation beginning with the shortest step and followed by taller and taller rows with the tallest step stopping last. Thus, to obtain a pattern of stereocilia in rows of increasing height what transpires are progressive go signals followed by a period when all the stereocilia grow and ending with progressive stop signals. We discuss how such a sequence could be controlled.
An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some... more An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some filaments are surrounded by a set of three windings that form a sheath. We provide evidence that the sheath is composed of subunits having a molecular mass of 24,000 Da. We suggest that the sheath could be composed of protofilaments of flagellin wound around the filament.
Using a new procedure that combines electron-density correlation with biochemical information, we... more Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium.
The size of the putative export channel in the bacterial flagellar filament appears small (25 A) ... more The size of the putative export channel in the bacterial flagellar filament appears small (25 A) in studies done by electron microscopy but large (60 A) in studies done by X-ray diffraction. We have undertaken additional studies by electron microscopy to examine some of the possible causes of the difference. A comparison of three-dimensional image reconstructions of native and reconstituted filaments rules out the presence or absence of flagellin monomers in the export channel as the source of the variation in apparent channel size. The channel seen in reconstructions from both kinds of filaments is 25 A in diameter. The difference in the previous studies is more probably a result of artifacts introduced in either the X-ray or the electron microscopical methodology. Comparisons of three-dimensional reconstructions from images of filaments embedded in various stains (anionic, cationic and neutral) and in ice, taken at a range of defocuses, rule out the two most likely sources of artifact in electron microscopy (i.e., staining artifacts and defocus phase contrast). Based on these studies we suggest that the channel seen in the image reconstructions is free of exported flagellin monomers, that its true diameter is about 25 A, and, therefore, that the flagellin monomer must be unfolded to pass along it.
Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signalin... more Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin-fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin-fimbrin bundles. Furthermore, the assembly of the actin-fimbrin arrays suggests coupling between actin polymerization, fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity.
Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are appr... more Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822-834) on the packing of actin filaments in this stereocilia.
Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin fil... more Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.
We have determined the structure of the actin-scruin filament to 13-A resolution using a combinat... more We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)-ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross-links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.
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