From Static Images To Dynamic Structures: The Use of Image Analysis and Video In Deriving Dynamical Information From Electron Microscopy

Advances in computer graphics and numerical processing, in video technology, and in image acquisition have enabled us to extend the power of the electron microscope in the analysis of macromolecular structures, particularly helical protein polymers. Three applications of this technology will be described:1) There are frequently times where the static images acquired from fixed, stained or frozen specimens leads to a loss of information about the dynamical properties of the molecules or structures being studied. We have been using computed image analysis, graphics and animation to recover the dynamical information that can be obtained from electron microscopic images.Using the RecA protein of E. coli , we have been able to capture different biochemical states as a function of time through the use of a slowly hydrolyzable ATP analog, ATP-γ-S. Threedimensional reconstruction of these helical structures, combined with computer-generated animation between different structures, have enabled us to directly visualize the motions within the protein polymer associated with the hydrolysis of the nucleotide analog. Modifications of the RecA protein, achieved through either proteolysis or mutation, have allowed us to use the same techniques to visualize domain-domain movements within the RecA filament which occur over a range of 5-10Å. The methods of analysis, graphics and animation which have been used will be discussed. The general applicability of these procedures to other systems will also be addressed.