Assembly of Acanthamoeba myosin-II minifilaments.: Model of anti-parallel dimers based on EM and X-ray diffraction of 2D and 3D crystals

Current models suggest that the first step in the assembly of Acanthamoeba myosin-II is anti-parallel dimerization of the coiled-coil tails with an overlap of 15 nm. Sedimentation equilibrium experiments showed that a construct containing the last 15 heptads and the non-helical tailpiece of the myosin-II tail (15T) forms dimers. To examine the structure of the 15T dimer, we grew 3D and 2D crystals suitable for X-ray diffraction and electron image analysis, respectively. For both conditions, crystals formed in related space and plane groups with similar unit cells (a = 87.7 Angstrom, b = 64.8 Angstrom, c = 114.9 Angstrom, beta = 108.0degrees). Inspection of the X-ray diffraction pattern and molecular replacement analysis revealed the orientation of the coiled-coils in the unit cell. A 3D density map at 15 Angstrom in-plane resolution derived from a tilt series of electron micrographs established the solvent content of the 3D crystals (75%, v/v), placed the coiled-coil molecules at the approximate translation in the unit cell, and revealed the symmetry relationships between molecules. On the basis of the low-resolution 31) structure, biochemical constraints, and X-ray diffraction data, we propose a model for myosin interactions in the anti-parallel dimer of coiled-coils that guide the first step of myosin-II assembly. (C) 2004 Elsevier Ltd. All rights reserved.