Supporting Information

Supporting Protocol S1. Protein crystallization, data collection and structure refinement.

Protein crystal soaking experiments

Typically, 0.5 mL of soak solution containing 10mM MgCl2 was added first to the crystal drop, followed a few seconds later with 1 mL of soak solution containing 200 mM DNA (hairpin or linear 5’-AATCT-3’), 25mM ATP (lithium salt, Sigma) and/or 25 mM GTP (lithium salt, Sigma) or 25mM b,g-methyleneadenosine 5’-triphosphate (AMPPCP, lithium salt, Sigma) and 20% (v/v) glycerol. The solutions were left to diffuse through the crystal for 1-10 min or until cracking was observed. A dataset was also collected for the E634Q apoenzyme without any soaking.

Data collection and processing

X-ray diffraction data for WT f6 RdRp complex structures (WT-Tri4T-ATP-Mg2+, WT-5’-AATCT-3’-ATP-GTP-Mg2+, WT-5’-AATCT-3’-GTP-Mg2+) were collected on station I03 at the Diamond Synchrotron, Oxfordshire, UK using an ADSC Q315 3x3 CCD detector. Data for E634Q apoenzyme and different complexes (E634Q-hairpin-AMPPCP-Mg2+) were collected on station ID14-EH2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France using an ADSC Q4R CCD detector. Each image covered an oscillation of 1o and exposure times were typically 1-2 or 45-60 seconds for WT and E634Q complexes, respectively. All data were collected at liquid nitrogen temperatures after soaking the crystals for 1-10 min in the specified condition in the presence of 20% (v/v) glycerol as cryoprotectant. Data for E634Q apoenzyme was collected after briefly washing the crystals with glycerol. WT-Tri4T-ATP-Mg2+ and WT-5’-AATCT-3’-GTP-Mg2+ data were scaled and indexed using HKL-2000 (4), but for WT-5’-AATCT-3’-ATP-GTP-Mg2+, the E634Q apoenzyme and E634Q complexes the automated data reduction program XIA2 (6) with the 3D option (using Labelit, XDS and XSCALE algorithms) (3) was used.

Structure refinement

For WT-Tri4T-ATP-Mg2+, visual inspection of the SIGMAA 2|Fo|-|Fc| difference-Fourier maps in COOT (2) allowed modelling of two ATP molecules at the active site of all three molecules. In molecules I and III, a Mg2+ could be built at the non-catalytic ion site. In molecule II, a Mg2+ could be built between the two phosphate backbones of the bound ATPs, and additionally four nucleotides of DNA hairpin (5’-AATG-3’) could be fitted at the template entrance reaching over a positively charged groove on the outer surface of the polymerase stabilised by the crystal packing of nearby molecule III a-helix.

For WT-5’-AATCT-3’-ATP-GTP-Mg2+ visual inspection of the SIGMAA 2|Fo|-|Fc| difference-Fourier maps in COOT (2) allowed modelling of GTP and the triphosphate backbone of an ATP at the active site in all three molecules. A Mg2+ ion could be built at the non-catalytic ion site in all molecules. Part of the 5’-AATCT-3’ DNA soaked into the crystal could be fitted at the entrance to the template tunnel in the same position seen for WT-Tri4T-ATP-Mg2+. Due to local disorder of the more external nucleotides, only 1 or 2 nucleotides of DNA were built for molecules I and III respectively. For molecule II, 4 nucleotides of DNA (5’-AATC-3’) could be built convincingly into the stronger positive difference density, stabilised by a nearby molecule III a-helix.

For WT-5’-AATCT-3’-GTP-Mg2+ visual inspection of the SIGMAA 2|Fo|-|Fc| difference-Fourier maps in COOT (2) allowed modelling of a single GTP at the active site and a Mg2+ at the non-catalytic ion site in all three molecules of the crystal asymmetric unit. No evidence for DNA was seen for molecule I and only two nucleotides of DNA were built for molecule III (5’-TC-3’) presumably due to local disorder of the more external nucleotides. For molecule II, four nucleotides of DNA (5’-AATC-3’) could be built into the stronger positive difference density, stabilised by a nearby molecule III a-helix.

E634Q hairpin complexes were determined by rigid body refinement of the three molecules present in the ASU with PHENIX (1), using the WT apo P21 structure as a starting model. Visual inspection of the SIGMAA 2|Fo|-|Fc| difference-Fourier maps for the hairpin complexes in COOT (2) allowed modelling of the triphosphates of an AMPPCP molecule bound to all three monomers of the ASU and a Mg2+ ion bound only in molecules I and II at the non-catalytic ion site. Part of the template could also be modelled in the template tunnel, with 3, 4 and 6 nucleotides modelled for molecules I, II and III respectively. The positions where AMPPCP, Mg2+ and DNA oligonucleotides could be traced into electron density were consistent across all four E634Q complexes. A second Mg2+ ion could be built in molecule I of E634Q-Tri4T-AMPPCP-Mg2+ and E634Q-Tetra2T-AMPPCP-Mg2+ structures, co-ordinated by the triphosphates of AMPPCP, the side chain of A345 and backbone carbonyl group of A344. In molecule III, residues 606-614 and 631-647 are highly disordered in all four E634Q hairpin complexes, with no continuous peptide density for these regions in the |Fo|-|Fc| maps; these residues were removed for subsequent refinement steps.

For the E634Q apostructure (no soaking) visual inspection of the initial model and manual rebuilding was carried out in COOT (2). A Mn2+ ion could be built at the non-catalytic ion site for all three monomers.

Clear differences in the strength and position of the electron density in the DNA and NTP binding regions were detectable for all WT and E634Q complexes. Therefore ligands observed in each molecule present in the ASU were modelled and refined separately. All refinements were as described in the main text, see Table 2 for detailed statistics.

Fig. S1. Thermal denaturation curves of f6 WT, E491Q and E634Q RdRps.

The thermal shift assay (ThermoFluor) was carried out in a real-time PCR machine (BioRad DNA Engine Opticon 2) where buffered solutions of protein (WT, E491Q and E634Q RdRps) and fluorophore (SYPRO Orange; Molecular Probes, Invitrogen), with and without additives, were heated in a stepwise fashion from 25oC to 95oC. 5mL protein (1 mg/mL) and 5 mL SYPRO Orange (Molecular Probes, Invitrogen) were made up to a total assay volume of 50 mL with reference buffer (50 mM Tris–HCl, pH 8.0, 50 mM NaCl) in white low profile thin-wall PCR plates (Abgene) sealed with microseal ‘B’ films (BioRad). The fluorophore was excited in the range of 470–505 nm and fluorescence emission was measured in the range of 540–700 nm every 0.5oC after a 10 s hold. The additives screened were: 0.1 mM, 1 mM or 5 mM EDTA; 5 mM EDTA with 10 mM MgCl2; 5 mM EDTA with 10 mM MnCl2; 10 mM MgCl2 without EDTA; 10 mM MnCl2 without EDTA. Denaturation curves for these conditions are shown for (A) WT, (B) E491Q and (C) E634Q polymerases, and are coloured according to the legend. Close-ups for each condition (indicated by black boxes) are provided to distinguish curves from one another about the melting temperature (Tm). Black arrows indicate the shift of curves to higher or lower Tm values in accordance with the text. The Tm values were calculated by applying a Boltzmann Distribution Equation about the sigmoidal melting curves to obtain the inflection point of the slope (the midpoint of the unfolding transition), using the GraphPad Prism software and are shown in Table 1 of the paper with standard errors.

Fig. S2. Electron density map for WT crystals soaked in EDTA, at the structural Mn ion binding site.

Data were collected from a crystal of WT polymerase soaked in 50mM EDTA. The data were collected on ID14-EH2 ( l = 0.933 Å). The sigma weighted 2Fo-Fc map, is contoured at 1.8s, and coloured blue. An anomalous fourier map is displayed in green, contoured at 3 s, showing that manganese is no longer bound at the non-catalytic site. This model and data have been deposited with PDB code XXXXX.

Table S1. Purification and crystallization conditions for selected viral RdRps with non-catalytic divalent ions*.

Virus / PDB ID / Published ion / Purification and storage conditions / Divalent ions in crystallization conditions / Comments
West Nile virus / 2hcs / No ion / Protease inhibitor tablet (EDTA) / - / -
2hcn / Ca2+ / 200 mM Ca-acetate / From map and coordination geometry it is unlikely to be Ca2+
2hfz / Mg2+ / 300 mM MgCl2 / Map and coordination geometry consistent with Mg2+
Foot-and-mouth disease virus / 1u09 / No ion / 1 mM EDTA / - / -
1wne / Mg2+ / 2 mM MgCl2
200 mM Mg-acetate / Structure factors not available
Rabbit hemorrhagic disease virus / 1khv / (H2O) / 0.25 mM EDTA
10 mM MgCl2 / 5 mM LuCl3 / Lu3+ ion 6Å from Mn2+ in f6. Evidence for Mg2+ in place of H2O at Mn2+ site
1khw / Density difficult to interpret / 5 mM LuCl3 +
50 mM Mg(NO3)2,18 mM MnCl2 / Poor density in region of active site makes interpretation difficult
Norwalk virus / 1sh0,1sh2 / No ion / 0.25 mM EDTA / - / -
1sh3 / Mg2+ / 20 mM MgSO4 / Map and coordination geometry consistent with Mg2+
Dengue virus / 2j7u / Mg2+ / 1 mM EDTA / 1 mM EDTA, up to 200 mM MgSO4 or Mg-acetate / Map and coordination geometry consistent with Mg2+
Poliovirus / 1rdr / Ca2+ / 0.1 mM EDTA / 0.2 mM EDTA, 600-1200 mM CaCl2 / From map and coordination geometry it is unlikely to be Ca2+
Reovirus / 1n35 / No ion / 1 mM EDTA / - / -
1mwh / Mn2+ / 1.5 mM Mn2+ / Cap bound structure with Mn2+ ion at equivalent site
Qβ / 3mmp / No ion / 1 mM EDTA, 5 mM MgCl2/20 – 250 mM imidazole, 5 mM MgCl2 / -
3agq / Mg2+ / No information available / 200 mM Mg(OAc)2 / Map consistent with Mg2+
3agp / Ca2+ / No information available / 200 mM Ca(OAc)2 / Map consistent with Ca2+

*The table is an extended version of that reported in ref. (5). The new columns are highlighted by a strong black border. Recent data on Qβ RdRp is also included.

Supplementary References

1. Adams, P. D., R. W. Grosse-Kunstleve, L. W. Hung, T. R. Ioerger, A. J. McCoy, N. W. Moriarty, R. J. Read, J. C. Sacchettini, N. K. Sauter, and T. C. Terwilliger. 2002. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58:1948-54.

2. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126-32.

3. Kabsch, W. 1993. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. Journal of Applied Crystallography 26:795-800.

4. Otwinowski, Z. 1997. Processing of X-ray diffraction data collected in the oscillation mode. Meth Enzymol 276:307-326.

5. Poranen, M. M., P. S. Salgado, M. R. Koivunen, S. Wright, D. H. Bamford, D. I. Stuart, and J. M. Grimes. 2008. Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase. Nucleic Acids Res 36:6633-44.

6. Winter, G. 2010. xia2: an expert system for macromolecular crystallography data reduction. J Appl Cryst 43:186-190.