Supplementary Information. Structural basis for duplex RNA recognition and cleavage by A.

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1 Supplementary Information Structural asis for duplex RNA recognition and cleavage y A. fulgidus C3PO Eneida arizotto 1, Edward D Lowe 1 & James S Parker 1 1 Department of Biochemistry University of Oxford Oxford United Kingdom Correspondence should e addressed to J.S.P. (james.parker@ioch.ox.ac.uk)

2 Supplementary Figure 1. Structure-ased sequence alignment of AfTrax and representative eukaryotic Trax and Translin proteins. AfTrax secondary structure (helices α1 α6 and loops) and sequence numering are shown across the top. Dotted regions are asent from the structure. Red downward triangles indicate conserved catalytic glutamate or aspartate residues. The residue mutated in AfTrax-mut (D114A) is highlighted y a red star. Diamonds indicate RNA-interacting residues; squares indicate residues whose side chains interact with RNA-interacting residues. Yellow (diamonds or squares) indicates residues from Trax-like suunits, whereas purple indicates residues from Translin-like suunits. The eukaryotic sequences are from humans (h), zerafish (z), Drosophila (d), S. pome (sp) and A. thaliana (At). Shading shows sequence conservation in either the Trax or Translin sufamily (oth including the AfTrax sequence.) The figure was formatted using ALINE 54.

3 2 3 Rotation plus (right-handed) superhelical shift 1 4 Highest suunit Lowest suunit (Trax-like suunit) Tyr181 (in each suunit) Internal cavity c d e N α1 α3 N AfTrax Drosophila Trax Human Trax N Human Translin ouse Translin Drosophila Translin AfTrax α4 α2 α6 α5 C C C Supplementary Figure 2. AfC3PO octamer and monomer structure. (a) Octameric suunit arrangement in AfC3PO. In this view of the apo structure, the four suunits of the upper tetramer are colored yellow, green, lue and magenta and the four suunits of the lower tetramer, in a similar ut inverted arrangement, are colored uniformly grey. Catalytic residues from each of the eight suunits are colored red and laeled 1 8. () Superhelical suunit arrangement in AfC3PO. Suunits in the upper and lower tetramers are related y a ~ 9 rotation and a small right-handed superhelical shift along the rotation axis. This creates a shallow spiral arrangement, with one suunit (yellow) adopting the lowest position in the spiral and another (shown in green) the highest. Tyr181 side chains are depicted (red) to illustrate the relative shift along the rotation axis. (c) Superposition of the four (independent) chains that make up either the upper or lower AfC3PO tetrameric unit, colored red, yellow, green and lue. Inter-suunit Cα root mean square deviations (rmsds) are.8.89 Å. α helices α1 α6 of the fold are laeled. (d) Superposition of a representative AfTrax monomer with Trax from humans 17 (3PJA/J, rmsd 1.95 Å) and Drosophila 3 (3RIU/C, rmsd 2.32 Å). (e) Superposition of a representative AfTrax monomer with Translin from humans 17 (3PJA/A, rmsd 2.31 Å), mouse 31 (1KEY/A, rmsd 2.42 Å) and Drosophila 3 (3RIU/A, rmsd 2.45 Å).

4 A28 asorance (mau) µ AfTrax (D114A) 1 µ duplex RNA 5 µ duplex RNA 2.5 µ duplex RNA 1 µ duplex RNA 1 µ AfTrax (D114A) + 1 µ duplex RNA 1 µ AfTrax (D114A) + 5 µ duplex RNA 1 µ AfTrax (D114A) µ duplex RNA 1 µ AfTrax (D114A) + 1 µ duplex RNA AfTrax (D114A) RNA complexes ( 4) Free RNA 1 AfTrax (D114A) in isolation Elution positions of molecular weight markers (kda) Elution volume (ml) RNA DNA * * C C A G A C A C A C A A C C A C U U C G A C G C G U C G A A U U C C A G A C A C A C A A C C A C T T C G A C G C G T C G A A T T *P P P P P P P P P P P P P P P P *P P P P P P P P P P P P P P P P *P P P P P P P P P P P P P P P P *P P P P P P P P P P P P P P P P 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ 1 μ Complexes Free nucleic acids Supplementary Figure 3. Interactions of AfTrax (D114A) with nucleic acids. (a) Analytical gel filtration profiles of AfTrax (D114A) in isolation and in complex with various concentrations of 14 p sirna-like RNA duplex (duplex as crystallized). The AfTrax concentration (1 µ) refers to the AfTrax monomer concentration. The elution positions of standard molecular weight markers are shown across the ottom. Wild type AfTrax elutes at the same position as AfTrax (D114A) (not shown). () Native gel moility shift assays showing AfTrax (D114A) association with laelled (*) 16 mer RNA and DNA, single-stranded and doule-stranded.

5 Ideal A-form RNA P14 Strand 2 P3 P4 P4 P13 P2 P5 P5 P1 P6 P6 P7 P7 Strand 1 P1 P9 P8 P8 Deviation from ideal A-form RNA (Å) Strand 1 Strand 2 P3 P13 P2 P1 P1 P9. P1 P2 P3 P4 P5 P6 P7 P8 P9 P1 P13 P14 Phosphate from end P14 c d Riose C1ʹ Riose C1ʹ Phosphate Phosphate Separation (Å) Base pair Supplementary Figure 4. Conformation of duplex RNA ound inside AfC3PO. (a) Superposition of an ideal A-form RNA duplex (green) with the RNA duplex ound inside AfC3PO (strands red and lue; cartoon representation). Phosphate groups (P1 P14) are laeled from the ends. () Chart depicting deviation from A-form structure for the phosphate groups on the two strands. (c) View of the duplex illustrating inter-strand phosphate and riose C1ʹ distances (dotted pink and green respectively). Base pairs are numered 1 13 from one end. (d) Chart depicting inter-strand distances along the duplex. Base pairs are numered as in panel c. Terminal phosphate groups (1 and 14) and ase pairs (1 and 13) show increased separations.

6 P1 2.6 Å 2.2 Å 2.1 Å 2.1 Å W W P9 P1 Supplementary Figure 5. Electron density at the AfC3PO Trax-like suunit catalytic sites. (a) 2Fo Fc electron density contoured at 1. σ surrounding the sustrate RNA, catalytic metal () and coordinating conserved catalytic glutamate side chains. W is a coordinated water. The putative scissile ond is indicated y a red arrow. etal coordination distances are shown. () Fo Fc difference density contoured at 3. σ around one strand of the sustrate RNA and a Trax-like suunit catalytic site. The putative scissile ond is indicated y a red arrow.

7 Human Translin AfC3PO Translin -like suunit (No adjacent suunit in truncated Drosophila C3PO) AfC3PO Translin -like suunit Asp168 Asp196 Lys199 Asp168 Lys166 Lys242 Arg171 Lys166 Lys253 Arg171 Ala/Asp114 Asp193 Glu8 Glu126 Glu129 Glu197 Arg121 Arg2 His39 His75 Ala/Asp114 Asp24 Glu8 Glu123 Glu126 Arg121 Arg211 His39 His7 Glu28 Human Trax AfC3PO Trax -like suunit Drosophila Trax AfC3PO Trax -like suunit c d e AfC3PO Trax-like suunits AfC3PO Translin-like suunits ( 6) Human Trax Human Translin ( 6) Drosophila Trax (truncated suunits) Drosophila Translin ( 4) (truncated suunit) Supplementary Figure 6. Comparisons of AfC3PO, human C3PO and truncated Drosophila C3PO. (a) Superposition of an AfC3PO Trax-like suunit catalytic site (RNA omitted) with a Trax catalytic site from human C3PO 17 (PDB code 3QB5, chains K and C). AfC3PO suunits are colored orange and grey, with yellow side chains, and human suunits are colored purple (Trax) and lue (Translin) with purple side chains. A manganese ion (, purple sphere) soaked into crystals of human C3PO occupies the same coordinated position as the magnesium ion (, green sphere ehind) in the RNA complex of AfC3PO. () Superposition of an AfC3PO Trax-like suunit catalytic site (RNA omitted) with a Trax catalytic site from truncated Drosophila C3PO 3 (PDB code 3RIU, chain C), formed from truncated Trax and Translin suunits. Coloring is as in panel a, with Drosophila replacing human. There is no similar adjacent Translin suunit in the truncated Drosophila C3PO crystal structure. (c) AfC3PO octamer with ound RNA. (d) Human C3PO octamer (PDB code 3PJA) 17 composed of two Trax suunits (salmon and violet) and six Translin suunits (lue). (e) Truncated Drosophila C3PO hexamer (PDB code 3RIU) 3 composed of two truncated Trax suunits (salmon and violet) and four truncated Translin suunits (lue).