(A) Schematic showing the functional domains of human Dicer. Crystal structures representative of most functional domains are known. Green checks indicate successful localization of the indicated positions using DOLORS. Red crosses indicate failed attempts. (B) A representative view of the full-length Dicer EM map with major features labeled. (C) C-terminal AviTag Dicer and control wild type Dicer were incubated with BirA in a biotinylation reaction. The streptavidin-labeled Dicer co-eluted with His₆-streptavidin from a His Trap column. The BirA enzyme, containing a His₆-tag, co-eluted from the column. (*) Dicer contains a protease sensitive loop in the RNase III domains that when cleaved does not significantly alter the structure of the protein. EM micrographs of (D) unlabeled human Dicer and (E) C-terminal streptavidin-labeled Dicer. The lower panels display representative class averages from the two datasets.

Streptavidin was attached to a loop in either (A) PAZ or (C) Platform domain of Dicer. 2D class averages of labeled proteins are shown with corresponding 3D RCT reconstructions. Each map is overlaid onto the unlabeled Dicer EM map (shown in gray). The estimated attachment sites for streptavidin are indicated by spheres (PAZ: Blue, Platform: Green). The estimated attachment sites for (B) PAZ-labeled and (D) Platfrom-labeled Dicer from 8 independent RCT reconstructions mapped onto the unlabeled Dicer EM map. (E) Crystal structures of PAZ (Blue) and Platform (green) domains with the AviTag insertion sites indicated by spheres. The distance between the two sites is 40 Å. (F) The estimated attachment sites for PAZ and Platform are separated by an average distance of 40 Å. (G) PAZ-Platform crystal structure modeled into the EM map based on labeling results.

The RNase IIIb domain was labeled by attaching streptavidin to either (A) a 45 amino acid residue surface loop or (B) the same loop truncated to 25 residues. 2D class averages are shown with corresponding 3D RCT reconstructions overlaid on the unlabeled Dicer EM map, shown in gray (left panels). The estimated streptavidin attachment sites are indicated by red spheres. Estimated attachment sites from 8 RCT reconstructions mapped on the Dicer EM map (right panels). Dashed circles indicate the smallest sphere in which all sites fall. (C) The streptavidin density (colored mesh) from 8 independent RCT reconstructions from C-terminal, PAZ, Platform, and RNase III labeled Dicer, overlaid onto the EM map of Dicer (solid gray). The C-terminal attachment sites are diffuse and difficult to localize. The PAZ and Platform attachment sites are tightly clustered, allowing a positional estimate within a 10-Å radius. The RNase IIIb attachment site, while more flexible than those of PAZ and Platform, still allowed a positional estimate within a 20Å radius.

Dicer was labeled within the loop prior to (A) (DUF-prior) and after (B) (DUF-after) the DUF283 domain. 2D class averages are shown with corresponding 3D RCT reconstructions. Each map is overlaid onto the unlabeled Dicer EM map (shown in gray). The estimated attachment sites for streptavidin are indicated by spheres (DUF-prior: pink and DUF-after: cyan). (C) Positions of streptavidin attachment sites for DUF-prior and DUF-after follow the proposed path of the polypeptide, from helicase to PAZ/Platform, within Dicer EM map.