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 A, b=64.8 A, c=114.9 A, beta=108.0 degrees). 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 15A 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 3D 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.
Turbedsky
Kirsi
K
Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
Pollard
Thomas D
TD
Yeager
Mark
M
D000048:Acanthamoeba / Q000378:metabolism*; D000818:Animals; D018360:Crystallography, X-Ray; D019281:Dimerization; D004583:Electrons; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D046529:Microscopy, Electron, Transmission; D024462:Myosin Type II / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry; D011487:Protein Conformation; D014961:X-Ray Diffraction; D014965:X-Rays
D024462:Myosin Type II; D009218:Myosins