content top

Thin Filament Detail

Thin Filament Detail

4 info needed

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 average of originally extracted repeats prior to subvolume alignment. From left to right are a central section of the initial global average, a surface view in the same direction as the original tomogram and a surface view from a direction along the inter-thick-filament axis showing the paired bulges due to troponin. Since this is a stained specimen, regions of protein (stain rich and thus low electron translucency) are dark in this representation (low electron counts) and regions rich in embedding medium, and thus high electron translucency are bright (high electron counts). (B) Global average after subvolume alignment is completed. The 2.75 nm stagger on different sides of the actin filament is now visible as is the different spacing separating troponin densities on opposite sides of the actin filament. The negative stain effect in the specimen is now pronounced. In the surface view, middle, both the troponin densities and the myosin heads have increased definition. The appearance suggests the presence of four myosin heads, two on each actin long pitch helical strand. Viewed along the filament axis, the difference in spacing of the troponin densities on the front and back surfaces (left, red, and right, blue, in this view) is easily visible. From Wu et al., 2009, J Struct Biol 168: 485-502.

Read More

Myosin Head Fitting

Myosin Head Fitting

need more info

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 color for easier identification. From Wu et al., 2009, J Struct Biol 168: 485-502.

Read More

Myosin-Actin Attachments

Myosin-Actin Attachments

Myosin-Actin Attachments

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 is built in. The number in the upper right is the number of the corresponding raw repeat subvolume from among the 515 total subvolumes. Small panels to the left are the central section and an opaque isodensity surface view of the larger panel without the quasiatomic model. Actin long pitch strands are cyan and green with the four target-zone actins, which can accept strong binding myosin attachments, in darker shades, TM is yellow and Tn orange. Strongly bound myosin heads are red, weak binding myosin heads are magenta, The essential light chain is dark blue and the regulatory light chain light blue. (A) shows a single headed cross-bridge on the left and a 2-headed, strong binding cross-bridge on the right. (B) shows a pair of 1-headed, strong-binding cross-bridges on actin subunits H and I. (C & D) have a 2-headed cross-bridge on the left and a 1-headed cross-bridge on the right, all strongly bound to actin. (E & F) are mask motifs with Tn-bridges. In (E) the right side M-ward weak binding cross-bridge is bound outside of the target zone to TM near actin subunit F while the one on the left is within the target zone on actin subunit I. In (F), the weak binding, left-side, M-ward cross-bridge is bound outside the target zone to TM near actin subunit G while the weak binding cross-bridge on the right is bound to target-zone actin subunit H. Tn-bridges have not been fit with a myosin head. These six reassembled repeats can also be viewed in Supporting Movies S1-S6. From Wu et al., 2010, PLoS-ONE

Read More

Insect Flight Muscle

Insect Flight Muscle

still awaiting

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 computed. To obtain the thin filaments and their crossbridges, a reassembled class average of each original raw repeat was placed into an idealized lattice with appropriate rotations and translations and then low pass filtered to the original resolution of the tomogram. The two maps were then displayed in Chimera, the column average of thick filaments colored yellow and the reassembled thin filaments colored gray. Myosin head attachments to the troponin complex were subsequently colored green in Adobe Photoshop.

Read More

Welcome to the Taylor Lab

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.

Read More
content top