Structure of the RNA-dependent RNA polymerase P2 from the cystovirus φ8

Figures

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  Fig. 1. Sequence alignment of P2 RdRps from bacteriophages φ8, φ6 and φ12.The alignment was generated using Clustal Omega46. Sequence annotation was generated using the ESPript web service47. Conserved residues are highlighted in red while similar residues have a red-type colour and are surrounded by a blue outline. The pink asterisks indicate the residues interacting with metal ions. The secondary structure assignment for φ8 P2 is shown on top of the sequence and the fingers, palm, thumb and C-terminal subdomains coloured in red, green, blue and yellow respectively.

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  Fig. 2. Structure of φ8 P2 and AF2 predictions. (a) Cartoon representation of the crystal structure of φ8 P2 showing the fingers, palm, thumb and C-terminal subdomains coloured in red, green, blue and yellow respectively. The bound Ca2+ ion is shown as a magenta sphere. (b) Comparison of the refined φ8 P2 model coloured in yellow and the five AF2 predicted models coloured in grey. The first 50 residues of N-terminal subdomains of each model are coloured in red to highlight the structural differences.

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  Fig. 3. X-ray structures of cystoviral RdRps. (a) The crystal structures of φ8 P2, φ6 P2 (PDB: 1HHS) and φ12 P2 (PDB: 4GZK) proteins are shown in cartoon representation with the fingers, palm, thumb and C-terminal subdomains coloured in red, green, blue and yellow respectively. The pink asterisks indicate the location of the β-hairpin found in φ8 P2 and φ6 P2. (b) Comparison of the N-terminal of φ8 P2, φ6 P2 and φ12 P2. The first 50 residues of φ8 P2 and equivalent residues in φ6 P2 and φ12 P2 are coloured in red while the rest of the proteins are coloured in grey. (c) ssRNA binding site of φ6 P2 on the left (PDB: 1UVJ) and φ8 P2 on the right. Proteins are shown in cartoon representation and side chains and RNA in sticks. Oxygen, nitrogen and phosphorous atoms are coloured in red, blue and orange respectively. Carbon atoms from φ6 P2, RNA and φ8 P2 are coloured in pink, silver and grey respectively.

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  Fig. 4. Metal ion binding sites in cystoviral RdRps. (a) Non-catalytic ion binding site of φ8 P2. Ca2+ is shown as a magenta sphere and protein residues as sticks. 2fo-fc electron density is contoured at 1 σ and shown as transparent surface. (b) Non-catalytic and catalytic metal ion binding sites of φ8 P2 (carbon coloured in yellow, top left), φ12 P2 (carbon coloured in grey, top right, PDB: 4IEG), φ6 P2 (carbon coloured in cyan, bottom left, PDB code: 1HI0; bottom right, PDB code: 1UVN)). Calcium, manganese and magnesium ions are shown as spheres and coloured in magenta, grey and brown respectively.

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  Fig. 5. Fitting of φ8 P2 in the φ6 procapsid cryo-EM reconstruction. Localised reconstruction of φ6 P2 in the procapsid at 7.9Å resolution (EMD-3185) fitted with the X-ray crystal structures of φ8 P2 coloured as in Fig. 2a, and φ8 P1 (PDB code: 4BX4) coloured in dark orange. The pink asterisks indicate the location of the β-hairpin.