Thin filament detail within the global average of repeat subvolumes from an electron tomogram of isometrically contracting insect flight muscle after alignment and averaging. The specimen used for the tomography has been smash frozen against a liquid helium cooled copper block, freeze substituted, embedded, sectioned and stained. (A) Global...
Fitting of weak and strong binding myosin heads. Color rendered fit of a repeat containing two weak binding myosin heads (magenta colored heavy chain) and one strong binding myosin head (red colored heavy chain). The TM strand is colored yellow, Tn orange, ELC blue, RLC cyan. Target zone actins are in a darker shade of their actin strand...
Diversity of myosin-actin attachments. These reconstructions obtained by subvolume classification and averaging of a specimen of insect flight muscle fast frozen by smashing into a liquid He cooled copper block, freeze substituted, embedded, sectioned and stained. Each repeat reassembled from up to 6 class averages before the atomic model...
Portion of an electron tomogram assembled from rebuilt class averages of isometrically contracting insect flight muscle. To make this picture, the thick filaments were segmented separately from the aligned raw repeats and then placed back into an idealized lattice with appropriate rotations and translations. A column average was then...
Our laboratory is using 3-D electron microscopy (3DEM) to determine the structures of proteins and macromolecular assemblages in muscle and the cytoskeleton. Although we use most 3-D imaging methods, our primary imaging method is electron tomography. Our approaches are somewhat from the norm in that we use exclusively marker-free image alignment. We are also at the forefront in the application of correspondence analysis to volume data from electron tomography.
Our longest running 3-D reconstruction project investigates the structure of myosin crossbridges in different states using the highly ordered filament lattice of insect flight muscle (IFM). In concert with the structural studies, we are developing 3-D reconstruction algorithms uniquely suitable for studying muscle structure.
We are also developing methods for analyzing the structure of paracrystalline specimens. This research is an outgrowth of our studies on IFM structure but the technology is applicable to many paracrystalline specimens. One of these techniques is a unique tomographic reconstruction method that uses crosscorrelation methods to align the images in a tilt series. To deal with the specimen disorder, which is manifest as variations in crossbridge structure, we are extending the widely used methods of 2-D correspondence analysis to 3-D motifs obtained by tomography. Finally, the knowledge of the atomic structure of the two important proteins in muscle contraction, myosin and actin, provides a unique opportunity to extend the low resolution information obtained by 3DEM to atomic resolution. The first and most important step in electron crystallography is the formation of 2-D crystalline arrays, which are the most suitable specimen for this technique. The first protein that we have successfully crystallized by this method is α-actinin. We have also formed 2-D arrays of smooth muscle HMM, smooth muscle myosin, and myosin-V.
As well as 2D protein crystallization, we utilize lipid monolayers to assemble multiprotein complexes in 2-D paracrystalline arrays. These arrays make structural analysis easier because they remove superposition problems that complicate image interpretation. The methodology for formation of what we call 2-D bundles open a number of avenues for research into the structure of the cytoskeleton.
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