Refinement of the F-Actin Model against X-ray Fiber Diffraction Data by the Use of a Directed Mutation Algorithm

Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin

“…The position arrived at for phalloidin was the one giving the lowest R-factor, rather than one determined on the basis of difference Fourier maps. Thus, as the actual electron density for phalloidin was not obtained by Lorenz et al [39] its positioning relative to the filament axis must be regarded as tentative.…”

“…However, in both cases the cross-linker was too long to pinpoint the phalloidin binding site. Lorenz et al [39], using a`directed mutation algorithm', placed the phalloidin site at the intersection of three actin subunits in the Holmes±Lorenz model of F-actin, close to the fiber axis. However, there are doubts about this analysis.…”

“…It was also concluded that an F-actin model based on the actin ribbon seen in the profilin:b-actin crystal could not account for the position of the gold particles. However, in modelling the position of gold-labelled phalloidin on the side of the monomer in the ribbon-derived filament opposite that expected from mutagenesis data [41], it was assumed that the position of phalloidin relative to the fiber axis had been determined unambiguosly by Lorenz et al [39] (see earlier). If instead the gold-labelled phalloidin had been placed over the phosphate-binding loops near G158, R177 and D179, in conformance with the mutagenesis data on yeast actin [10], both the cross-linking results and the position of the gold label could be accounted for with models resulting from the ribbonto-helix conjecture, including the one presented [41].…”

An actin point‐mutation neighboring the ‘hydrophobic plug’ causes defects in the maintenance of cell polaroty and septum organization in the fission yeast Schizosaccharomyces pombe

“…Actin might function via interaction with a variety of its binding proteins, depending on the cell cycle stages. However, their structure/function relationships as well as the regulatory mechanisms of the interactions are poorly understood, although the three-dimensional structure of the actin molecule has been progressively refined in recent years [8][9][10][11]. To solve such problems, comparative analyses between changes in structure of functional molecules and in vivo phenotypes caused by mutations might be useful.…”

Single-particle reconstruction from EM images of helical filaments

“…On the other hand, it has been known that due to the cylindrical averaging and overlap of different Bessel functions in x-ray fiber diffraction, models derived from this technique may not be unique, either. For example, a fiber diffraction model for F-actin [31] provided "an almost perfect fit" to the observed x-ray diffraction pattern, but has subsequently been shown to have little experimental support [32]. Hopefully, higher resolution EM reconstructions of such filamentous phages as fd will be able to resolve this controversy generated by the different models from NMR, x-ray fiber diffraction and EM.…”