Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction

Area of Science:

• Structural Biology
• Biophysics
• Electron Microscopy

Background:

• X-ray crystallography is a primary method for determining protein structures.
• Cryoelectron microscopy (cryo-EM) offers an alternative imaging technique for biological macromolecules.

Purpose of the Study:

• To compare the structural information obtained from cryo-EM images with existing x-ray diffraction data of tropomyosin crystals.
• To assess the potential of cryo-EM for resolving structural features not easily discernible via x-ray methods.

Main Methods:

• Comparison of computed transforms from cryo-EM images with x-ray diffraction data.
• Correction of cryo-EM images for lattice distortions and contrast transfer function.
• Constraining structure factors to the plane group symmetry (pmg) of the projection.
• Analysis of amplitude and phase data from multiple cryo-EM images against 3D x-ray diffraction data.

Main Results:

• Cryo-EM data achieved a resolution of approximately 18 Å, comparable to x-ray crystallography's 15 Å resolution.
• The average R factor between cryo-EM and x-ray amplitudes was 15%, with a mean phase difference of 4.8 degrees.
• Cryo-EM density maps revealed structural features like filament width, run, and crossover region appearance, consistent with x-ray data.

Conclusions:

• Cryo-EM provides a reliable method for structural analysis of tropomyosin, yielding results comparable to x-ray crystallography.
• Preliminary studies on frozen-hydrated tropomyosin/troponin cocrystals indicate cryo-EM's potential for uncovering novel structural details.