Structural Determination of Macromolecular Machines Guided By Proteomics and Electron Microscopy

Abstract

Models of macromolecular assemblies are essential for a mechanistic description of cellular processes. Low-resolution density maps of these assemblies are increasingly obtained by electron-microscopy techniques. In addition, interactions between subunits in these assemblies can be systematically mapped by proteomics techniques.We have developed MultiFit [1], a method used for simultaneously fitting atomic structures of components into their assembly density map at resolutions as low as 25 A. The method was benchmarked on large assemblies of known structures. It generally finds a near-native configuration in one of the 10 top scoring solutions. The component positions and orientations are optimized with respect to a scoring function that includes the quality-of-fit of components in the map, the protrusion of components from the map envelope, as well as the shape complementarity between pairs of components. The scoring function is optimized by our exact inference optimizer DOMINO that efficiently finds the global minimum in a discrete sampling space. DOMINO decomposes the set of optimized variables into relatively uncoupled but potentially overlapping subsets that can be sampled independently form each other, followed by efficiently gathering the subset solutions into the global minimum.We have further extended MultiFit for modeling the architecture of macromolecular assemblies by aligning proteomics data into electron-microscopy density maps. The method facilitated the structural modeling of the AAA-ATPase/20S core particle sub-complex of the 26S proteasome [2].[1] K. Lasker, M. Topf, A. Sali, H. Wolfson. Inferential optimization for simultaneous fitting of multiple components into a cryoEM map of their assembly. Journal of Molecular Biology 388, 180-194, 2009.[2] F. Forster, K. Lasker, F. Beck, S. Nickell, A. Sali, W. Baumeister. An Atomic Model AAA-ATPase/20S core particle sub-complex of the 26S proteasome. Biochem Biophys Res Commun 388, 228-233, 2009