The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
John Trinick - One of the best experts on this subject based on the ideXlab platform.
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Negative Stain em of myosn s1 bound to actin and thin filaments in mgatp after rapid mixing
Biophysical Journal, 2014Co-Authors: Howard D White, Matt L Walker, Betty Belknap, John TrinickAbstract:We have used rapid mixing Negative Stain electron microscopy (EM) to observe myosin heads (S1) bound to F-actin and thin filaments in the presence of ATP. Myosin V S1 was mixed with excess ATP, incubated for 2 sec to allow binding and hydrolysis, and then mixed with either F-actin or cardiac thin filaments. This mixture was applied to an EM grid and Negatively Stained with uranyl acetate ∼20 ms later. Short times on the grid allow protein concentrations as high as 10 uM to be used, which is critical to observe weakly binding complexes, such as S1-ADP-Pi with actin filaments. This approach has advantages compared to hand mixed solutions. It has improved time resolution (0.3 s vs 3 s between mixing and Staining), the mixing is ∼1000x faster compared to manual manipulation (∼ 2 ms vs 2 s) and mixing is more uniform. The entire automated procedure used an apparatus with computer controlled stepper motors previously built for time-resolved cryo-EM (White et al, J Struct Biol:144, 246-52, 2003), which was modified for Negative Staining. Slow and non-uniform mixing produced by hand mixing can suggest cooperative binding. After automated mixing, S1-ADP-Pi with cardiac thin filaments at low calcium concentrations, S1 bound randomly and only rarely were bare or fully decorated filaments observed. Without ATP, thin filaments were mostly either bare or fully decorated, suggestive of cooperativity. These results may explain the apparently contradictory observations that binding of S1 to thin filaments appeared to be cooperative in micrographs, whereas kinetic and structural measurements indicate that individual rigor heads activate 12-14 actin subunits.
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use of Negative Stain and single particle image processing to explore dynamic properties of flexible macromolecules
Journal of Structural Biology, 2004Co-Authors: Stan A Burgess, Matt L Walker, John Trinick, Kavitha Thirumurugan, Peter J KnightAbstract:Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unStained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used Negative Staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of Negative Staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the Stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.
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flexibility within myosin heads revealed by Negative Stain and single particle analysis
Journal of Cell Biology, 1997Co-Authors: S A Burgess, Matt L Walker, Howard D White, John TrinickAbstract:Electron microscopy of Negatively Stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of ∼2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in Staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating Negative Staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at myosinhead.html). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.
Harold P Erickson - One of the best experts on this subject based on the ideXlab platform.
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Negative Stain electron microscopy of inside out ftsz rings reconstituted on artificial membrane tubules show ribbons of protofilaments
Biophysical Journal, 2012Co-Authors: Sara L Milam, Masaki Osawa, Harold P EricksonAbstract:FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by Negative-Stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for Negative-Stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.
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imaging ftsz rings in vitro by Negative Stain em
Biophysical Journal, 2012Co-Authors: Sara L Milam, Masaki Osawa, Harold P EricksonAbstract:In prokaryotes, the major cytoskeleton protein involved in cell division is FtsZ, a homolog of tubulin. In vivo, light microscopy studies have shown that FtsZ forms a ring (Z ring) at midcell during cell division. However, the substructure of the Z ring remains a mystery. In vitro, FtsZ assembles into short, single-stranded protofilaments (pfs), but it is unknown how these 120 nm long pfs come together to form a ring that encompasses the ∼3000 nm circumference of the cell. One possibility is that they associate by lateral contacts to make ribbons of protofilaments. Another is that the pfs are not in direct contact but communicate via distortions of the membrane. We have developed a lipid tubule system that has enabled us to image the Z-ring substructure by Negative-Stain EM. Here we took advantage of the self-assembling lipid, DC 8,9, PC, which spontaneously forms 500 nm diameter tubules in solution. We added membrane-targeted FtsZ (mts-FtsZ), which has been shown to form Z rings inside or outside of tubular liposomes. The mts-FtsZ assembled characteristic Z rings imaged by fluorescence light microscopy. Negative-Stain EM showed that these Z rings consist of ribbons of pfs, which were one-subunit thick. Fourier transform analysis of these images confirmed the close packing (∼5 nm) of individual pfs in the Z ring. Overall, the data suggest that lateral contacts are important for Z ring assembly. In addition, these results were similar for FtsZ from E. coli and M. tuberculosis, suggesting that lateral contacts are an intrinsic property of Z rings.
Matt L Walker - One of the best experts on this subject based on the ideXlab platform.
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Negative Stain em of myosn s1 bound to actin and thin filaments in mgatp after rapid mixing
Biophysical Journal, 2014Co-Authors: Howard D White, Matt L Walker, Betty Belknap, John TrinickAbstract:We have used rapid mixing Negative Stain electron microscopy (EM) to observe myosin heads (S1) bound to F-actin and thin filaments in the presence of ATP. Myosin V S1 was mixed with excess ATP, incubated for 2 sec to allow binding and hydrolysis, and then mixed with either F-actin or cardiac thin filaments. This mixture was applied to an EM grid and Negatively Stained with uranyl acetate ∼20 ms later. Short times on the grid allow protein concentrations as high as 10 uM to be used, which is critical to observe weakly binding complexes, such as S1-ADP-Pi with actin filaments. This approach has advantages compared to hand mixed solutions. It has improved time resolution (0.3 s vs 3 s between mixing and Staining), the mixing is ∼1000x faster compared to manual manipulation (∼ 2 ms vs 2 s) and mixing is more uniform. The entire automated procedure used an apparatus with computer controlled stepper motors previously built for time-resolved cryo-EM (White et al, J Struct Biol:144, 246-52, 2003), which was modified for Negative Staining. Slow and non-uniform mixing produced by hand mixing can suggest cooperative binding. After automated mixing, S1-ADP-Pi with cardiac thin filaments at low calcium concentrations, S1 bound randomly and only rarely were bare or fully decorated filaments observed. Without ATP, thin filaments were mostly either bare or fully decorated, suggestive of cooperativity. These results may explain the apparently contradictory observations that binding of S1 to thin filaments appeared to be cooperative in micrographs, whereas kinetic and structural measurements indicate that individual rigor heads activate 12-14 actin subunits.
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use of Negative Stain and single particle image processing to explore dynamic properties of flexible macromolecules
Journal of Structural Biology, 2004Co-Authors: Stan A Burgess, Matt L Walker, John Trinick, Kavitha Thirumurugan, Peter J KnightAbstract:Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unStained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used Negative Staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of Negative Staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the Stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.
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the structure of dynein c by Negative Stain electron microscopy
Journal of Structural Biology, 2004Co-Authors: Stan A Burgess, Matt L Walker, Hitoshi Sakakibara, Kazuhiro Oiwa, Peter J KnightAbstract:Abstract Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in Negative Stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the microtubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of the structure of dynein.
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flexibility within myosin heads revealed by Negative Stain and single particle analysis
Journal of Cell Biology, 1997Co-Authors: S A Burgess, Matt L Walker, Howard D White, John TrinickAbstract:Electron microscopy of Negatively Stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of ∼2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in Staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating Negative Staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at myosinhead.html). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.
Sara L Milam - One of the best experts on this subject based on the ideXlab platform.
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Negative Stain electron microscopy of inside out ftsz rings reconstituted on artificial membrane tubules show ribbons of protofilaments
Biophysical Journal, 2012Co-Authors: Sara L Milam, Masaki Osawa, Harold P EricksonAbstract:FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by Negative-Stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for Negative-Stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.
-
imaging ftsz rings in vitro by Negative Stain em
Biophysical Journal, 2012Co-Authors: Sara L Milam, Masaki Osawa, Harold P EricksonAbstract:In prokaryotes, the major cytoskeleton protein involved in cell division is FtsZ, a homolog of tubulin. In vivo, light microscopy studies have shown that FtsZ forms a ring (Z ring) at midcell during cell division. However, the substructure of the Z ring remains a mystery. In vitro, FtsZ assembles into short, single-stranded protofilaments (pfs), but it is unknown how these 120 nm long pfs come together to form a ring that encompasses the ∼3000 nm circumference of the cell. One possibility is that they associate by lateral contacts to make ribbons of protofilaments. Another is that the pfs are not in direct contact but communicate via distortions of the membrane. We have developed a lipid tubule system that has enabled us to image the Z-ring substructure by Negative-Stain EM. Here we took advantage of the self-assembling lipid, DC 8,9, PC, which spontaneously forms 500 nm diameter tubules in solution. We added membrane-targeted FtsZ (mts-FtsZ), which has been shown to form Z rings inside or outside of tubular liposomes. The mts-FtsZ assembled characteristic Z rings imaged by fluorescence light microscopy. Negative-Stain EM showed that these Z rings consist of ribbons of pfs, which were one-subunit thick. Fourier transform analysis of these images confirmed the close packing (∼5 nm) of individual pfs in the Z ring. Overall, the data suggest that lateral contacts are important for Z ring assembly. In addition, these results were similar for FtsZ from E. coli and M. tuberculosis, suggesting that lateral contacts are an intrinsic property of Z rings.
Peter J Knight - One of the best experts on this subject based on the ideXlab platform.
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use of Negative Stain and single particle image processing to explore dynamic properties of flexible macromolecules
Journal of Structural Biology, 2004Co-Authors: Stan A Burgess, Matt L Walker, John Trinick, Kavitha Thirumurugan, Peter J KnightAbstract:Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unStained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used Negative Staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of Negative Staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the Stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.
-
the structure of dynein c by Negative Stain electron microscopy
Journal of Structural Biology, 2004Co-Authors: Stan A Burgess, Matt L Walker, Hitoshi Sakakibara, Kazuhiro Oiwa, Peter J KnightAbstract:Abstract Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in Negative Stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the microtubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of the structure of dynein.
