The Experts below are selected from a list of 52056 Experts worldwide ranked by ideXlab platform
Radostin Danev - One of the best experts on this subject based on the ideXlab platform.
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Using the Volta phase plate with defocus for cryo-EM Single Particle Analysis.
eLife, 2017Co-Authors: Radostin Danev, Dimitry Tegunov, Wolfgang BaumeisterAbstract:Previously, we reported an in-focus data acquisition method for cryo-EM Single-Particle Analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 A.
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Using the Volta phase plate with defocus for cryo-EM Single Particle Analysis
2016Co-Authors: Radostin Danev, Dimitry Tegunov, Wolfgang BaumeisterAbstract:Previously, we reported an in-focus data acquisition method for cryo-EM Single Particle Analysis with the Volta phase plate (VPP) (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also enables 3D reconstructions with resolutions in the 2 A range, demonstrating that, in practice, there are no resolution limitations imposed by the VPP.
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cryo em Single Particle Analysis with the volta phase plate
eLife, 2016Co-Authors: Radostin Danev, Wolfgang BaumeisterAbstract:One way of investigating how proteins and other biological molecules work is to look at their structure. Light microscopes cannot produce detailed enough images to fully reveal these structures, and so a technique called cryo-electron microscopy is often used instead. In this technique, a biological sample is frozen to the temperature of liquid nitrogen and a beam of electrons is fired at it to create an image. By taking many of these images and then subjecting them to computer processing it is possible to reconstruct the three-dimensional structure of the molecule. Frozen biological samples are essentially transparent to the electron beam used in an electron microscope. To view samples, researchers therefore use a method called phase contrast, which relies on a property of the electron beam (called its phase) changing as the beam passes through the sample. The traditional “defocus” method of producing phase contrast from electron microscopy relies on processing a series of slightly out-of-focus images of the sample. Phase plates are add-on devices that are commonly used in light microscopes to produce phase contrast. For many years now, attempts have been made to produce a working phase plate for electron microscopes. However, an effective plate, called the Volta phase plate, has only recently been developed. Danev and Baumeister have now evaluated how well the Volta phase plate performs during the Analysis of a Single, relatively large protein. This molecule is considered ‘easy’ to analyze using cryo-electron microscopy as relatively few microscopic images need to be recorded to solve the protein’s structure. Danev and Baumeister found that the Volta phase plate matched or slightly exceeded the performance of the traditional defocus method of producing phase contrast, depending on how many images were used to analyze the protein. This is the first time that a phase plate has matched the performance of the defocus method. A future challenge will be to make the experimental procedures and the software involved in using the Volta phase plate more user-friendly. The phase plate also needs to be tested with more ‘difficult’ samples, such as small proteins and samples whose structure could not be established using the defocus method of producing phase contrast.
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Automated Cryo-tomography and Single Particle Analysis with a New Type of Phase Plate
Microscopy and Microanalysis, 2014Co-Authors: Radostin Danev, Bart Buijsse, Yoshiyuki Fukuda, Maryam Khoshouei, Juergen M. Plitzko, Wolfgang BaumeisterAbstract:Recent years have shown an increased interest in the development and use of phase plates in cryo-EM. The oldest and the most productive type of phase plate is the carbon film Zernike phase plate [1]. It has been successfully used in cryo-tomography [2] and Single Particle Analysis applications [3]. Despite its good performance the Zernike phase plate has a few pitfalls. One major practical hindrance is its short lifetime [4]. Typically within 10 days after being installed into the microscope its performance deteriorates to the point where it has to be exchanged. Another disadvantage of the Zernike phase plate is that it produces fringes around high-contrast features in the image, such as lipid membranes, support film edges etc [5]. Despite its shortcomings the Zernike phase plate has been the main motivation and experience generator in the last years.
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Single Particle Analysis based on zernike phase contrast transmission electron microscopy
Journal of Structural Biology, 2008Co-Authors: Radostin Danev, Kuniaki NagayamaAbstract:We present the first application of Zernike phase-contrast transmission electron microscopy to Single-Particle 3D reconstruction of a protein, using GroEL chaperonin as the test specimen. We evaluated the performance of the technique by comparing 3D models derived from Zernike phase contrast imaging, with models from conventional underfocus phase contrast imaging. The same resolution, about 12 A, was achieved by both imaging methods. The reconstruction based on Zernike phase contrast data required about 30% fewer Particles. The advantages and prospects of each technique are discussed.
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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Using the Volta phase plate with defocus for cryo-EM Single Particle Analysis.
eLife, 2017Co-Authors: Radostin Danev, Dimitry Tegunov, Wolfgang BaumeisterAbstract:Previously, we reported an in-focus data acquisition method for cryo-EM Single-Particle Analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 A.
-
Using the Volta phase plate with defocus for cryo-EM Single Particle Analysis
2016Co-Authors: Radostin Danev, Dimitry Tegunov, Wolfgang BaumeisterAbstract:Previously, we reported an in-focus data acquisition method for cryo-EM Single Particle Analysis with the Volta phase plate (VPP) (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also enables 3D reconstructions with resolutions in the 2 A range, demonstrating that, in practice, there are no resolution limitations imposed by the VPP.
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cryo em Single Particle Analysis with the volta phase plate
eLife, 2016Co-Authors: Radostin Danev, Wolfgang BaumeisterAbstract:One way of investigating how proteins and other biological molecules work is to look at their structure. Light microscopes cannot produce detailed enough images to fully reveal these structures, and so a technique called cryo-electron microscopy is often used instead. In this technique, a biological sample is frozen to the temperature of liquid nitrogen and a beam of electrons is fired at it to create an image. By taking many of these images and then subjecting them to computer processing it is possible to reconstruct the three-dimensional structure of the molecule. Frozen biological samples are essentially transparent to the electron beam used in an electron microscope. To view samples, researchers therefore use a method called phase contrast, which relies on a property of the electron beam (called its phase) changing as the beam passes through the sample. The traditional “defocus” method of producing phase contrast from electron microscopy relies on processing a series of slightly out-of-focus images of the sample. Phase plates are add-on devices that are commonly used in light microscopes to produce phase contrast. For many years now, attempts have been made to produce a working phase plate for electron microscopes. However, an effective plate, called the Volta phase plate, has only recently been developed. Danev and Baumeister have now evaluated how well the Volta phase plate performs during the Analysis of a Single, relatively large protein. This molecule is considered ‘easy’ to analyze using cryo-electron microscopy as relatively few microscopic images need to be recorded to solve the protein’s structure. Danev and Baumeister found that the Volta phase plate matched or slightly exceeded the performance of the traditional defocus method of producing phase contrast, depending on how many images were used to analyze the protein. This is the first time that a phase plate has matched the performance of the defocus method. A future challenge will be to make the experimental procedures and the software involved in using the Volta phase plate more user-friendly. The phase plate also needs to be tested with more ‘difficult’ samples, such as small proteins and samples whose structure could not be established using the defocus method of producing phase contrast.
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Automated Cryo-tomography and Single Particle Analysis with a New Type of Phase Plate
Microscopy and Microanalysis, 2014Co-Authors: Radostin Danev, Bart Buijsse, Yoshiyuki Fukuda, Maryam Khoshouei, Juergen M. Plitzko, Wolfgang BaumeisterAbstract:Recent years have shown an increased interest in the development and use of phase plates in cryo-EM. The oldest and the most productive type of phase plate is the carbon film Zernike phase plate [1]. It has been successfully used in cryo-tomography [2] and Single Particle Analysis applications [3]. Despite its good performance the Zernike phase plate has a few pitfalls. One major practical hindrance is its short lifetime [4]. Typically within 10 days after being installed into the microscope its performance deteriorates to the point where it has to be exchanged. Another disadvantage of the Zernike phase plate is that it produces fringes around high-contrast features in the image, such as lipid membranes, support film edges etc [5]. Despite its shortcomings the Zernike phase plate has been the main motivation and experience generator in the last years.
Edward P. Morris - One of the best experts on this subject based on the ideXlab platform.
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3-Dimensional Structure of Human Cardiac Muscle Myosin Filaments by Electron Microscopy and Single Particle Analysis
Biophysical Journal, 2012Co-Authors: Hind A. Al-khayat, John M. Squire, Robert W. Kensler, Steve Marston, Edward P. MorrisAbstract:In all muscles the interaction between actin and myosin filaments leads to muscle contraction and force production mediated by the hydrolysis of ATP. Actin filament structure is well understood to high resolution, but myosin filament structure is much less well defined. Myosin filaments are formed from complicated arrangements of myosin molecules and accessory proteins (e.g. C-protein [MyBP-C] and titin). We compare the arrangement of the myosin heads in the different species and how these arrangements change in diseased muscle. We have developed a method to solve the 3-dimensional (3D) structure of myosin filaments by Single Particle Analysis of electron microscope (EM) data. We already defined the 3D structure of myosin filaments of various muscles from different species by both X-ray diffraction modelling and EM and Single Particle Analysis including insect flight muscle, scallop striated muscle, fish skeletal muscle and rabbit cardiac muscle. We are now studying the 3D structure of myosin filaments isolated from human heart muscles.Mutations in cardiac myosin, C-protein and titin are known to be associated with cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand myosin-associated heart disease, it is important to understand the 3D structure of myosin filaments in normal heart muscle.Recently we have developed procedures to isolate human cardiac muscle myosin filaments preserving their highly ordered pseudo-helical structure thus making them amenable, for the first time, to EM and Single Particle image Analysis. We have collected EM data from myosin filaments isolated from both normal and failing hearts, and have so far processed the data from normal heart muscle. Analysis of the 3D structure of myosin filaments in normal heart muscle will permit the structural effects of known myosin filaments-associated mutations to be investigated in detail.
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Three-dimensional structure of the M-region (bare zone) of vertebrate striated muscle myosin filaments by Single-Particle Analysis.
Journal of molecular biology, 2010Co-Authors: Hind A. Al-khayat, Edward P. Morris, Robert W. Kensler, John M. SquireAbstract:The rods of anti-parallel myosin molecules overlap at the centre of bipolar myosin filaments to produce an M-region (bare zone) that is free of myosin heads. Beyond the M-region edges, myosin molecules aggregate in a parallel fashion to yield the bridge regions of the myosin filaments. Adjacent myosin filaments in striated muscle A-bands are cross-linked by the M-band. Vertebrate striated muscle myosin filaments have a 3-fold rotational symmetry around their long axes. In addition, at the centre of the M-region, there are three 2-fold axes perpendicular to the filament long axis, giving the whole filament dihedral 32-point group symmetry. Here we describe the three-dimensional structure obtained by a Single-Particle Analysis of the M-region of myosin filaments from goldfish skeletal muscle under relaxing conditions and as viewed in negative stain. This is the first Single-Particle reconstruction of isolated M-regions. The resulting three-dimensional reconstruction reveals details to about 55 A resolution of the density distribution in the five main nonmyosin densities in the M-band (M6′, M4′, M1, M4 and M6) and in the myosin head crowns (P1, P2 and P3) at the M-region edges. The outermost crowns in the reconstruction were identified specifically by their close similarity to the corresponding crown levels in our previously published bridge region reconstructions. The packing of myosin molecules into the M-region structure is discussed, and some unidentified densities are highlighted.
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Reference Free Single Particle Analysis Of Reconstituted Thin Filaments
Biophysical Journal, 2009Co-Authors: Danielle M. Paul, William Lehman, Alnoor Pirani, Roger Craig, Larry S. Tobacman, John M. Squire, Edward P. MorrisAbstract:A detailed three-dimensional structure of the muscle thin filament is required in order to understand its regulation. To this end we have applied a reference free Single Particle Analysis approach to electron microscope images of negatively stained reconstituted thin filaments from skeletal actin and cardiac tropomyosin and troponin. The filaments were prepared in a low Ca2+ buffer. For image Analysis the filaments were segmented into ∼800A long Particles centred on the troponin complex. Density attributable to troponin and tropomyosin is readily identifiable in the two-dimensional class averages and the three-dimensional reconstruction. The data have previously been analysed using a model-based Single Particle method (Pirani et al., 2005, 2006). Our non-model based approach and novel strand averaging procedure has enabled us to quantify directly the stagger or axial rise between adjacent troponin complexes (∼27.7A). Comparison with our previous Analysis of native thin filaments indicates that reconstituted filaments assemble with the same arrangement of troponin as in vivo, viz .in register on both helical strands with a ∼40 nm repeat. This indicates that troponin and tropomyosin can organise themselves on actin filaments without requiring any other sarcomeric proteins.Pirani A., Vinogradova M.V., Curmi P.M., King W.A., Fletterick R.J., Craig R., Tobacman L.S., Xu C., Hatch V., Lehman W. 2006. An atomic model of the thin filament in the relaxed and Ca2+-activated States. J Mol Biol 357(3):707-17.Pirani A., Xu C., Hatch V., Craig R., Tobacman L.S., Lehman W. 2005. Single Particle Analysis of relaxed and activated muscle thin filaments. J Mol Biol 346(3):761-72.
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3D structure of relaxed fish muscle myosin filaments by Single Particle Analysis.
Journal of structural biology, 2006Co-Authors: Hind A. Al-khayat, Edward P. Morris, Robert W. Kensler, John M. SquireAbstract:Abstract To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) Single Particle Analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head ‘crowns’ in the 429 A myosin filament repeat. The Analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60°, 60° and 0°, rather than the regular 40° characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous Analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated.
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Single Particle Analysis: A new approach to solving the 3D structure of myosin filaments
Journal of Muscle Research & Cell Motility, 2004Co-Authors: Hind A. Al-khayat, Edward P. Morris, John M. SquireAbstract:Knowledge of the structure of muscle myosin filaments is essential for a proper understanding of sarcomere structure and how myosin heads interact with the actin filaments to produce force and movement. Two principal methods have been used to define the myosin head arrays in filaments in the relaxed state, namely modelling from low-angle X-ray diffraction data and image processing of electron micrographs of isolated filaments. Analysis of filament images by 3D helical reconstruction, which imposes total helical symmetry on the structure, is very effective in some cases, but it relies on the existence of very highly ordered preparations of straight filaments. Resolutions achieved to date are about 70 Å. Modelling of X-ray diffraction data recorded from whole relaxed fish or insect muscles has also been used as an independent method. Although the resolution of the diffraction data is often also about 70 Å, the effective resolution of the modelling is very much higher than this because additional very high resolution data (e.g. from protein crystallography) is included in the Analysis. However, the X-ray diffraction method has to date provided only limited data on non-myosin thick filament proteins such as C-protein and titin and it cannot provide the polarity of the myosin head arrangement. Both the helical reconstruction and X-ray diffraction techniques have advantages and disadvantages, but their disadvantages are avoided in the new approach of Single Particle Analysis of electron micrograph data. Even using the same micrographs as for helical reconstruction, the resolution can be extended by this method to about 50 Å or better. In addition, it is not necessary to assume that the myosin filaments are helical; a significant advantage in the case of vertebrate myosin filaments where there is a known crossbridge perturbation. Here we describe the principles of all these approaches, but particularly that of Single Particle Analysis. We outline the application of Single Particle Analysis to myosin filaments from vertebrate skeletal and insect flight (IFM) muscle myosin filaments.
W. Sahle - One of the best experts on this subject based on the ideXlab platform.
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Single Particle Analysis of the accumulation mode aerosol over the northeast Amazonian tropical rain forest, Surinam, South America
Atmospheric Chemistry and Physics, 2005Co-Authors: R. Krejci, J. StrÖm, M. De Reus, W. SahleAbstract:Single Particle Analysis of aerosols Particles larger than 0.2 ?m diameter was performed on 24 samples collected over Surinam tropical rain forest and in the adjacent marine boundary layer (MBL) during the LBA-CLAIRE 98 campaign in March 1998. Elemental composition and morphology of 2308 Particles was determined using SEM-EDX. The aerosol Particles were divided into seven groups according to their chemical composition: organic Particles, mineral dust, aged mineral dust, sea salt, aged sea salt, Ca-rich, and biogenic aerosol. However the organic material in aerosol Particles cannot be identified directly by SEM-EDX, we present indirect method of detection of organic material using this technique. Samples were further divided with respect to the distinct atmospheric layers present in the tropical troposphere including MBL, continental mixed layer, cloud convective layer, free troposphere and region of deep convection outflow. The organic and mineral dust Particles are two major groups observed over the rainforest. In the MBL also sea salt Particles represented a large fraction between 15 and 27%. The organic Particles control much of the chemical characteristic of the aerosol in the continental tropical troposphere. Their abundance ranged from less than 20% in the MBL to more than 90% in the free troposphere between 4.5- and 12.6-km altitude. During the transport of the air masses from the MBL over the rain forest, fraction of organic aerosol Particles more than doubled, reaching 40?60% in the continental boundary layer. This increase was attributed to direct emissions of biogenic aerosols from the tropical vegetation. The high fraction of the organic accumulation mode Particles in the upper tropical troposphere could be a good indicator for the air masses originated over the tropical rain forest.
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Single Particle Analysis of the accumulation mode aerosol over the northeast Amazonian tropical rain forest, Surinam, South America
Atmospheric Chemistry and Physics Discussions, 2004Co-Authors: R. Krejci, J. StrÖm, M. De Reus, W. SahleAbstract:Single Particle Analysis of aerosols Particles larger than 0.2 µm diameter was performed on 24 samples collected over Surinam tropical rain forest and in the adjacent marine boundary layer (MBL) during the LBA-CLAIRE 98 campaign in March 1998. Elemental composition and morphology of 2308 Particles was determined using SEM-EDX. The aerosol Particles were divided into seven groups according to their chemical composition: organic Particles, mineral dust, aged mineral dust, sea salt, aged sea salt, Ca-rich, and biogenic aerosol. Samples were further divided with respect to the distinct atmospheric layers present in the tropical troposphere including MBL, continental mixed layer, cloud convective layer, free troposphere and region of deep convection outflow. The organic and mineral dust Particles are two major groups observed over the rainforest. In the MBL also sea salt Particles represented a large fraction between 15 and 27%. The organic Particles control much of the chemical characteristic of the aerosol in the continental tropical troposphere. Their abundance ranged from less than 20% in the MBL to more than 90% in the free troposphere between 4.5 and 12.6 km altitude. During the transport of the air masses from the MBL over the rain forest, fraction of organic aerosol Particles more than doubled, reaching 40-60% in the continental boundary layer. This increase was attributed to direct emissions of biogenic aerosols from the tropical vegetation. The high fraction of the organic accumulation mode Particles in the upper tropical troposphere could be a good indicator for the air masses originated over the tropical rain forest.
John M. Squire - One of the best experts on this subject based on the ideXlab platform.
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3-Dimensional Structure of Human Cardiac Muscle Myosin Filaments by Electron Microscopy and Single Particle Analysis
Biophysical Journal, 2012Co-Authors: Hind A. Al-khayat, John M. Squire, Robert W. Kensler, Steve Marston, Edward P. MorrisAbstract:In all muscles the interaction between actin and myosin filaments leads to muscle contraction and force production mediated by the hydrolysis of ATP. Actin filament structure is well understood to high resolution, but myosin filament structure is much less well defined. Myosin filaments are formed from complicated arrangements of myosin molecules and accessory proteins (e.g. C-protein [MyBP-C] and titin). We compare the arrangement of the myosin heads in the different species and how these arrangements change in diseased muscle. We have developed a method to solve the 3-dimensional (3D) structure of myosin filaments by Single Particle Analysis of electron microscope (EM) data. We already defined the 3D structure of myosin filaments of various muscles from different species by both X-ray diffraction modelling and EM and Single Particle Analysis including insect flight muscle, scallop striated muscle, fish skeletal muscle and rabbit cardiac muscle. We are now studying the 3D structure of myosin filaments isolated from human heart muscles.Mutations in cardiac myosin, C-protein and titin are known to be associated with cardiomyopathies (e.g. hypertrophic cardiomyopathy and dilated cardiomyopathy). In order to understand myosin-associated heart disease, it is important to understand the 3D structure of myosin filaments in normal heart muscle.Recently we have developed procedures to isolate human cardiac muscle myosin filaments preserving their highly ordered pseudo-helical structure thus making them amenable, for the first time, to EM and Single Particle image Analysis. We have collected EM data from myosin filaments isolated from both normal and failing hearts, and have so far processed the data from normal heart muscle. Analysis of the 3D structure of myosin filaments in normal heart muscle will permit the structural effects of known myosin filaments-associated mutations to be investigated in detail.
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Three-dimensional structure of the M-region (bare zone) of vertebrate striated muscle myosin filaments by Single-Particle Analysis.
Journal of molecular biology, 2010Co-Authors: Hind A. Al-khayat, Edward P. Morris, Robert W. Kensler, John M. SquireAbstract:The rods of anti-parallel myosin molecules overlap at the centre of bipolar myosin filaments to produce an M-region (bare zone) that is free of myosin heads. Beyond the M-region edges, myosin molecules aggregate in a parallel fashion to yield the bridge regions of the myosin filaments. Adjacent myosin filaments in striated muscle A-bands are cross-linked by the M-band. Vertebrate striated muscle myosin filaments have a 3-fold rotational symmetry around their long axes. In addition, at the centre of the M-region, there are three 2-fold axes perpendicular to the filament long axis, giving the whole filament dihedral 32-point group symmetry. Here we describe the three-dimensional structure obtained by a Single-Particle Analysis of the M-region of myosin filaments from goldfish skeletal muscle under relaxing conditions and as viewed in negative stain. This is the first Single-Particle reconstruction of isolated M-regions. The resulting three-dimensional reconstruction reveals details to about 55 A resolution of the density distribution in the five main nonmyosin densities in the M-band (M6′, M4′, M1, M4 and M6) and in the myosin head crowns (P1, P2 and P3) at the M-region edges. The outermost crowns in the reconstruction were identified specifically by their close similarity to the corresponding crown levels in our previously published bridge region reconstructions. The packing of myosin molecules into the M-region structure is discussed, and some unidentified densities are highlighted.
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Reference Free Single Particle Analysis Of Reconstituted Thin Filaments
Biophysical Journal, 2009Co-Authors: Danielle M. Paul, William Lehman, Alnoor Pirani, Roger Craig, Larry S. Tobacman, John M. Squire, Edward P. MorrisAbstract:A detailed three-dimensional structure of the muscle thin filament is required in order to understand its regulation. To this end we have applied a reference free Single Particle Analysis approach to electron microscope images of negatively stained reconstituted thin filaments from skeletal actin and cardiac tropomyosin and troponin. The filaments were prepared in a low Ca2+ buffer. For image Analysis the filaments were segmented into ∼800A long Particles centred on the troponin complex. Density attributable to troponin and tropomyosin is readily identifiable in the two-dimensional class averages and the three-dimensional reconstruction. The data have previously been analysed using a model-based Single Particle method (Pirani et al., 2005, 2006). Our non-model based approach and novel strand averaging procedure has enabled us to quantify directly the stagger or axial rise between adjacent troponin complexes (∼27.7A). Comparison with our previous Analysis of native thin filaments indicates that reconstituted filaments assemble with the same arrangement of troponin as in vivo, viz .in register on both helical strands with a ∼40 nm repeat. This indicates that troponin and tropomyosin can organise themselves on actin filaments without requiring any other sarcomeric proteins.Pirani A., Vinogradova M.V., Curmi P.M., King W.A., Fletterick R.J., Craig R., Tobacman L.S., Xu C., Hatch V., Lehman W. 2006. An atomic model of the thin filament in the relaxed and Ca2+-activated States. J Mol Biol 357(3):707-17.Pirani A., Xu C., Hatch V., Craig R., Tobacman L.S., Lehman W. 2005. Single Particle Analysis of relaxed and activated muscle thin filaments. J Mol Biol 346(3):761-72.
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3D structure of relaxed fish muscle myosin filaments by Single Particle Analysis.
Journal of structural biology, 2006Co-Authors: Hind A. Al-khayat, Edward P. Morris, Robert W. Kensler, John M. SquireAbstract:Abstract To understand the structural changes involved in the force-producing myosin cross-bridge cycle in vertebrate muscle it is necessary to know the arrangement and conformation of the myosin heads at the start of the cycle (i.e. the relaxed state). Myosin filaments isolated from goldfish muscle under relaxing conditions and viewed in negative stain by electron microscopy (EM) were divided into segments and subjected to three-dimensional (3D) Single Particle Analysis without imposing helical symmetry. This allowed the known systematic departure from helicity characteristic of vertebrate striated muscle myosin filaments to be preserved and visualised. The resulting 3D reconstruction reveals details to about 55 A resolution of the myosin head density distribution in the three non-equivalent head ‘crowns’ in the 429 A myosin filament repeat. The Analysis maintained the well-documented axial perturbations of the myosin head crowns and revealed substantial azimuthal perturbations between crowns with relatively little radial perturbation. Azimuthal rotations between crowns were approximately 60°, 60° and 0°, rather than the regular 40° characteristic of an unperturbed helix. The new density map correlates quite well with the head conformations analysed in other EM studies and in the relaxed fish muscle myosin filament structure modelled from X-ray fibre diffraction data. The reconstruction provides information on the polarity of the myosin head array in the A-band, important in understanding the geometry of the myosin head interaction with actin during the cross-bridge cycle, and supports a number of conclusions previously inferred by other methods. The observed azimuthal head perturbations are consistent with the X-ray modelling results from intact muscle, indicating that the observed perturbations are an intrinsic property of the myosin filaments and are not induced by the proximity of actin filaments in the muscle A-band lattice. Comparison of the axial density profile derived in this study with the axial density profile of the X-ray model of the fish myosin filaments which was restricted to contributions from the myosin heads allows the identification of a non-myosin density peak associated with the azimuthally perturbed head crown which can be interpreted as a possible location for C-protein or X-protein (MyBP-C or -X). This position for C-protein is also consistent with the C-zone interference function deduced from previous Analysis of the meridional X-ray pattern from frog muscle. It appears that, along with other functions, C-(X-) protein may have the role of slewing the heads of one crown so that they do not clash with the neighbouring actin filaments, but are readily available to interact with actin when the muscle is activated.
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Single Particle Analysis: A new approach to solving the 3D structure of myosin filaments
Journal of Muscle Research & Cell Motility, 2004Co-Authors: Hind A. Al-khayat, Edward P. Morris, John M. SquireAbstract:Knowledge of the structure of muscle myosin filaments is essential for a proper understanding of sarcomere structure and how myosin heads interact with the actin filaments to produce force and movement. Two principal methods have been used to define the myosin head arrays in filaments in the relaxed state, namely modelling from low-angle X-ray diffraction data and image processing of electron micrographs of isolated filaments. Analysis of filament images by 3D helical reconstruction, which imposes total helical symmetry on the structure, is very effective in some cases, but it relies on the existence of very highly ordered preparations of straight filaments. Resolutions achieved to date are about 70 Å. Modelling of X-ray diffraction data recorded from whole relaxed fish or insect muscles has also been used as an independent method. Although the resolution of the diffraction data is often also about 70 Å, the effective resolution of the modelling is very much higher than this because additional very high resolution data (e.g. from protein crystallography) is included in the Analysis. However, the X-ray diffraction method has to date provided only limited data on non-myosin thick filament proteins such as C-protein and titin and it cannot provide the polarity of the myosin head arrangement. Both the helical reconstruction and X-ray diffraction techniques have advantages and disadvantages, but their disadvantages are avoided in the new approach of Single Particle Analysis of electron micrograph data. Even using the same micrographs as for helical reconstruction, the resolution can be extended by this method to about 50 Å or better. In addition, it is not necessary to assume that the myosin filaments are helical; a significant advantage in the case of vertebrate myosin filaments where there is a known crossbridge perturbation. Here we describe the principles of all these approaches, but particularly that of Single Particle Analysis. We outline the application of Single Particle Analysis to myosin filaments from vertebrate skeletal and insect flight (IFM) muscle myosin filaments.