p19 and siRNA. a, Sequence alignment of p19 from the Tombusviridae family. The viruses with abbreviation and GenBank access numbers are as follows: tomato bushy stunt virus (TBSV) 979033; Cymbidium ringspot virus (CymRSV) 20087029; cucumber bulgarian virus (CBV) 30018257; maize necrotic streak virus (MNeSV) 10644292. Residues 27–158 from TBSV p19 were cloned and used in crystallization. Secondary structural elements in the X-ray structure are shown on the top. Dashed lines denote disordered residues. Conserved residues are coloured yellow. Residues directly contacting the siRNA duplex are marked by red triangles. b, The sequence of 21-nt siRNA used for crystallization. Residues contacting (direct and water-mediated) by the p19 homodimer are marked in red. c, Electrophoretic mobility shift assays for binding of p19 to RNA/DNA, with nucleic acid construct descriptions listed on the top. The bands in some lanes migrating faster than the duplex correspond to unannealed ssRNA. The majority of these ssRNAs form a duplex in the presence of p19 and are shifted to the bound RNA position. d, Electrophoretic mobility shift assays defining the length dependence of self-complementary siRNA duplexes for complex formation with p19. These self-complementary RNA sequences adopt either duplexes with 2-nt 3′-overhangs or alternate hairpin folds. The hairpin RNAs, which do not bind p19, are not shown.

p19–siRNA complex structure. a, A stereo view perpendicular to the two-fold axis. Individual monomers of the p19 dimer are coloured blue and magenta, while the two siRNA strands are coloured orange and pink. Two tryptophans from each monomer of the p19 dimer, which bracket the terminal base pairs at either end of the siRNA duplex, are shown in stick representation. b, c, Alternative mono views of the complex rotated by 90° along a different axis. d, RNA-contacting region (green) along the dimeric protein surface. e, Electrostatic surface of dimeric protein, with blue and red colours corresponding to positively and negatively charged patches.

p19 and siRNA structures in complex. a, Ribbon representation of the p19 dimer in the complex. The β-strands are labelled S1 to S4 and the α-helices are labelled H1 to H4. The symmetry-related blue and magenta p19 monomers are designated by unprimed and primed symbols. The dimer forms a continuous eight-stranded β-sheet, which cradles the siRNA duplex within its concave face. The ‘reading head’ helices H1 and H1′ extend from opposite edges of the β-sheet core. b, The side-chain interactions that position helix H1 relative to the β-sheet core of each monomer in the p19 dimer in the complex. c, siRNAs with opposite orientations in the complex, with colour-coding from blue to red, reflecting an increase in temperature factors. The RNA was oriented as in .

Details of p19–siRNA interactions. a, Stacking and hydrogen-bond/electrostatic recognition of RNA ends. b, Hydrogen-bonding/electrostatic recognition associated with the RNA major groove. Only one strand of the siRNA is shown for clarity. c, Hydrogen-bonding recognition associated with the RNA minor groove. Only the sugar–phosphate backbone is shown for clarity. The interacting protein side chains and RNA sugar–phosphate backbones are coloured as follows: oxygens in red, nitrogens in navy blue, phosphates in yellow, carbons from protein monomer I in green, and carbons from monomer II in cyan. Other colour schemes are as .