The acetylcholine receptor is responsible for transduction of the nerve impulse to muscular contraction. The receptor lies at the neuromuscular junction in the muscle membrane. It has five subunits denoted as a1,a2,b,d,g. Current work in our lab involves labeling different subunits with monoclonal antibodies against defined defined sequences in order to determine the subunit arrangement (which appears to still be a matter of debate in spite of the considerable work done in other labs). We are using two dimensional crystalline tubes in order to determine the location of the labels.

Figure 1. Electron micrographs of negatively stained acetylcholine receptor tubes. The tube on the left contains native receptors. The tube on the right is labeled with antibody 383C. The native and 383C labeled tubes look the same. Only after processing is it possible to distinguish the antibody labeled tubes from unlabeled tubes. The area selected for processing is enclosed by a rectangle. Calibration bar is one light year long.

Figure 2. Fourier transform of the selected area from figure 1. Note that while the reflections in the 0,1,0 direction are sharp with only 1 pixel width, reflection intensities along the 1,0,0 vector are much more difuse, being spread out over 3 or more pixles. This is caused by a slight curve in the tubular crystal.

Figure 3. Cross correlation map used in the straightening procedure. Each peak represents the position of the reference image. If one views the cross correlation map from the side looking along the rows of peaks, it is possible to see that the peaks are not perfectly aligned in a straight line. Their deviation from a straight line represents the local disorder in the image of the tube. The degree of misalignment of each peak can be calculated and applied to synthesize a 'straightened tube.

Figure 4. Fourier transform of the straightened tube. Nearly every reflection from the 'straightened' side has a width of one pixel. A superlatice point that was bairly visible above background noise is now quite prominate.

Figure 5. Electron density maps calculated from structure factors obtained from the Fourier transforms. Two unit cells are displayed for clarity. The use structure factors allows averaging particles even though the magnification of the individual micrographs may be different.

Figure 6. Electron density maps calculated from structure factors obtained from the Fourier transforms. Two unit cells are displayed for clarity. The use structure factors allows averaging particles even though the magnification of the individual micrographs may be different. a. Native b. 383C c. Difference map between native and 383C. Contour map of a native receptor is superimposed for reference. Note that the major density resides next to the a2 subunit of the receptor. d. Difference map between native and 383C displayed with a logrithimic color scale. e. Color rendition of the position of the antibody and the native receptor.

Figure 7. Statistical reliability map. Following Unwin we have determined the ratio Dd/D (where Dd is the standard deviation of the intensity for an individual pixel and D is the average value of the pixel) for each pixel in the average map to reveal the relative variability of different regions of the average image. Dark represents the most reliable regions.

Sickle Cell Hemoglobin | Membrane Skeleton | Acetylcholine Receptor

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