Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions.

Cryo-electron microscopy produces 3D density maps of molecular machines, which consist of various molecular components such as proteins and RNA. Segmentation of individual components in such maps is a challenging task, and is mostly accomplished interactively. We present an approach based on the immersive watershed method and grouping of the resulting regions using progressively smoothed maps. The method requires only three parameters: the segmentation threshold, a smoothing step size, and the number of smoothing steps. We first apply the method to maps generated from molecular structures and use a quantitative metric to measure the segmentation accuracy. The method does not attain perfect accuracy, however it produces single or small groups of regions that roughly match individual proteins or subunits. We also present two methods for fitting of structures into density maps, based on aligning the structures with single regions or small groups of regions. The first method aligns centers and principal axes, whereas the second aligns centers and then rotates the structure to find the best fit. We describe both interactive and automated ways of using these two methods. Finally, we show segmentation and fitting results for several experimentally-obtained density maps.

Stalled folding mutants in the triple beta-helix domain of the phage P22 tailspike adhesin.

The trimeric bacteriophage P22 tailspike adhesin exhibits a domain in which three extended strands intertwine, forming a single turn of a triple beta-helix. This domain contains a single hydrophobic core composed of residues contributed by each of the three sister polypeptide chains. The triple beta-helix functions as a molecular clamp, increasing the stability of this elongated structural protein. During folding of the tailspike protein, the last precursor before the native state is a partially folded trimeric intermediate called the protrimer. The transition from the protrimer to the native state results in a structure that is resistant to denaturation by heat, chemical denaturants, and proteases. Random mutations were made in the region encoding residues 540-548, where the sister chains begin to wrap around each other. From a set of 26 unique single amino acid substitutions, we characterized mutations at G546, N547, and I548 that retarded or blocked the protrimer to native trimer transition. In contrast, many non-conservative substitutions were tolerated at residues 540-544. Sucrose gradient analysis showed that protrimer-like mutants had reduced sedimentation, 8.0 S to 8.3 S versus 9.3 S for the native trimer. Mutants affected in the protrimer to native trimer transition were also destabilized in their native state. These data suggest that the folding of the triple beta-helix domain drives transition of the protrimer to the native state and is accompanied by a major rearrangement of polypeptide chains.